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Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota
Abstract
Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.
... In Ptr, the ToxA gene is present only in isolates that secrete the ToxA effector, and no homolog has been identified in non-producing isolates of Ptr [3]. ToxA has been found in Ptr and in other related species, whereas ToxB has been reported only in Ptr, but its inactive homologs, termed toxb, were identified in Ptr and other closely related and distantly related fungal species [7][8][9][10]. ...
... An independent origin of these effectors was suggested previously [7,8,12,14]. In Ptr, ToxA and ToxB were never localized to the same chromosome [12]. ...
... No clear evidence of ToxB horizontal transfer has been found to date, but homologs of ToxB are present in closely and distantly related species (e.g. P. bromi, Cochilobolus sativus, Alternaria alternata, Magnaporthe grisea) [8]. It has been suggested that ToxB was acquired vertically from a common ascomycete ancestor [8,15]. ...
In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements.
A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr’s adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr’s effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb ‘Starship’ transposon (dubbed ‘Horizon’) with a clearly defined target site and target site duplications. ‘Horizon’ was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative ‘Starship’ (dubbed ‘Icarus’) in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and ‘Icarus’ were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as ‘one-compartment’ based on calculated gene distances and evolutionary rates.
These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.
... It is in the order Pleosporales and family Pleosporaceae and is grouped with other agriculturally relevant species of Cochliobolus, Leptospheria, and Stagonospora Schoch et al. 2009) within the Pleosporales. Multi-gene phylogenetic analyses of sequence data of the MAT1-2 high-mobility group (HMG) box, glyceraldehyde phosphate dehydrogenase (GPD), and internal transcribed spacer (ITS) regions place Pyrenophora species in a monophyletic clade (Andrie et al. 2008;Zhang and Berbee 2001) and provide high confidence in Pyrenophora species designations (Andrie et al. 2008). ...
... It is in the order Pleosporales and family Pleosporaceae and is grouped with other agriculturally relevant species of Cochliobolus, Leptospheria, and Stagonospora Schoch et al. 2009) within the Pleosporales. Multi-gene phylogenetic analyses of sequence data of the MAT1-2 high-mobility group (HMG) box, glyceraldehyde phosphate dehydrogenase (GPD), and internal transcribed spacer (ITS) regions place Pyrenophora species in a monophyletic clade (Andrie et al. 2008;Zhang and Berbee 2001) and provide high confidence in Pyrenophora species designations (Andrie et al. 2008). ...
... Transcripts of the race 3 ToxB-like gene in Ptr have thus far only been detected in conidia (Strelkov et al. 2006). Isolates of the sister-species to Ptr, P. bromi, express ToxB-like genes (Pb ToxB) whose coding regions are *89 % similar to ToxB and are also often present in multi-copy; however, unlike ToxB, these sequences are variable, both within and between isolates (Andrie and Ciuffetti 2011;Andrie et al. 2008). Interestingly, heterologous expression of several Pb ToxB loci revealed that Pb ToxB proteins do not induce symptoms on their host grass species, B. inermis, but some will cause chlorosis on ToxBsensitive wheat cultivars (Andrie and Ciuffetti 2011). ...
Pyrenophora tritici-repentis (Ptr), causal agent of tan spot of wheat, is a necrotrophic fungus that presents an increasing threat to wheat production due to its rapid, global expansion. Despite its homothallic nature, Ptr populations have high genetic diversity, which positively impacts host range and virulence. Pathogenicity by Ptr is attributable to the production of host-selective toxins (HSTs) and follows an inverse gene-for-gene mechanism, in which HSTs are recognized by unique single dominant genes that confer both toxin-sensitivity and disease susceptibility. Studies addressing the mechanism of action of Ptr HSTs have unveiled both commonalities and complexities of the host response to these toxins. Resistance-like host responses triggered by the HSTs support the emerging hypothesis that necrotrophic pathogens exploit the host defense response as a mechanism to induce host cell death and ensure colonization. Recent advances in sequencing technology have facilitated the comparison of the genetic makeup of pathogenic and nonpathogenic isolates of Ptr. Such comparisons are providing insights into the genetic diversity of the pathogen and the mechanisms that dictate the increase in virulence and incidence of this important pathogen. Comparative genome analysis has also provided evidence that transposable elements (TEs) play a crucial role in genome re-arrangement and expansion, which contributes to the genomic flexibility to create and diversify effectors. © 2014 Springer-Verlag Berlin Heidelberg (outside the USA). All rights reserved.
... Homologs of ToxB also have been reported in other ascomycete fungi, including Pyrenophora bromi (Died.) Drechsler (Andrie et al., 2008). While the form of Ptr ToxB in race 4 isolates of P. tritici-repentis appears to have little or no chlorosisinducing activity in toxin-sensitive wheat (Figueroa Betts et al., 2011;Kim and Strelkov, 2007), Pb ToxB, the form of the toxin from P. bromi, does (Andrie and Ciuffetti, 2011). ...
... Unlike ToxA, which was shown to be acquired by P. tritici-repentis through horizontal gene transfer (Friesen et al., 2006), the distribution of ToxB among the different races of the fungus may reflect gene duplication, divergence, and loss (Andrie et al., 2008;Lamari et al., 2003). Silencing of the ToxB gene in a wild-type race 5 isolate of P. tritici-repentis revealed that Ptr ToxB is not only a pathogenicity factor essential for tan spot development, but that it also serves as a virulence factor contributing quantitatively to disease severity (Aboukhaddour et al., 2012). ...
The causal agent of tan spot of wheat, Pyrenophora tritici-repentis, is a necrotrophic ascomycete that produces multiple host-selective toxins (HSTs) responsible for the development of destructive foliar lesions. Ptr ToxB, a small proteinaceous HST produced by some races of P. tritici-repentis, causes extensive chlorosis on toxin-sensitive wheat genotypes. To investigate the role of Ptr ToxB in the pathogenicity and virulence of P. tritici-repentis, the open reading frame of the encoding gene, ToxB, was cloned from a wild-type race 5 isolate of the fungus and transferred into a Ptr ToxB nonproducing isolate of race 2. Ectopic integration of ToxB was confirmed by Southern blotting analysis with a digoxigenin (DIG)-labeled DNA probe, while the abundance of ToxB transcript in culture was evaluated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The production and secretion of Ptr ToxB were assessed by Western blotting analysis of the transformed fungal strains. The pathogenicity of the transformants was tested by inoculation on Ptr ToxB-sensitive and insensitive wheat genotypes. The Ptr ToxB-producing transformants were able to induce chlorosis in a host-specific manner only on the toxin-sensitive host genotype, resulting in significantly higher levels of disease than those caused by the nontransformed isolate. The extent of chlorosis was well correlated with the amount of Ptr ToxB produced. These results demonstrate that the acquisition of Ptr ToxB-producing ability is a sufficient condition for pathogenicity in P. tritici-repentis and confirm the role of this toxin in mediating the compatibility between the fungus and different genotypes of its wheat host.
... ToxB is synthesized as an 87-amino acid preprotein that upon cleavage of its secretory signal peptide results in a 64-amino acid mature protein. It appears to have a complex evolutionary history as suggested by the presence of homologs in non-pathogenic P. tritici-repentis isolates and several pathogenic ascomycetes (15). For example, some homologs found in isolates of Pyrenophora bromi (Pb ToxB) are not toxic to the P. bromi host, bromegrass, but are toxic to ToxB-sensitive wheat cultivars, whereas other homologs such as a naturally occurring variant of ToxB, Ptr toxb (toxb), found in some non-pathogenic P. tritici-repentis isolates have no toxic activity (16,17). ...
... A series of 1 H- 15 N HSQC experiments were recorded on lyophilized samples of 15 N-labeled ToxB or toxb dissolved in D 2 O. Amide protons were considered to be involved in hydrogen bonding if they were still visible after the first round of HSQC experiments. For each hydrogen bond, two distance restraints were applied for H N (i)-O(j) and N(i)-O(j). ...
... ToxB is synthesized as an 87-amino acid preprotein that upon cleavage of its secretory signal peptide results in a 64-amino acid mature protein. It appears to have a complex evolutionary history as suggested by the presence of homologs in non-pathogenic P. tritici-repentis isolates and several pathogenic ascomycetes (15). For example, some homologs found in isolates of Pyrenophora bromi (Pb ToxB) are not toxic to the P. bromi host, bromegrass, but are toxic to ToxB-sensitive wheat cultivars, whereas other homologs such as a naturally occurring variant of ToxB, Ptr toxb (toxb), found in some non-pathogenic P. tritici-repentis isolates have no toxic activity (16,17). ...
... A series of 1 H- 15 N HSQC experiments were recorded on lyophilized samples of 15 N-labeled ToxB or toxb dissolved in D 2 O. Amide protons were considered to be involved in hydrogen bonding if they were still visible after the first round of HSQC experiments. For each hydrogen bond, two distance restraints were applied for H N (i)-O(j) and N(i)-O(j). ...
Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective
toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein
with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures
of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a β-sandwich fold comprising three strands in each half that
are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent
β-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond,
higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose
that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of
activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus,
as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such
as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.
... ToxB, the gene coding for Ptr ToxB, is found in multiple copies in races that possess Ptr ToxB activity (Strelkov et al., 2002;Martinez et al., 2004;Aboukhaddour et al., 2009). Homologues of this gene have also been reported in several races that lack Ptr ToxB activity Martinez et al., 2004;Strelkov et al., 2006) and in other ascomycete fungi that are non-pathogenic on wheat (Andrie et al., 2008). Ptr ToxC also causes chlorosis, but on different host genotypes than Ptr ToxB. ...
... Similarly, D308, another race 3 isolate described earlier, lacked Ptr ToxB activity, but also carried a homologue of the ToxB gene (Strelkov et al., 2006). A ToxB homologue was also reported in avirulent race 4 isolates of the fungus and in other ascomycetes that are not pathogenic Downloaded by [University of Alberta] at 15:46 24 May 2013 on wheat (Strelkov et al., 2006;Andrie et al., 2008). The form of Ptr ToxB encoded by the race 4 isolates of P. tritici-repentis possesses little or no chlorosis-inducing activity (Kim & Strelkov, 2007;Figueroa Betts et al., 2011). ...
Tan spot is an important foliar disease of wheat caused by Pyrenophora tritici-repentis. Eight races of this fungus have been identified based on their virulence on a wheat differential set. Previous surveys revealed that most isolates collected from the Canadian Prairies belong to races 1 and 2 of P. tritici-repentis and carry the ToxA gene. However, pathogen populations from Alberta, a major wheat-producing province, have not been characterized. In this study, the virulence of 45 isolates of P. tritici-repentis from Alberta was evaluated on six differential wheat genotypes. Race 1 was found to be most common, representing 62% of the isolates tested, while race 2 was the second most common (36% of isolates). A single isolate of race 3 was also found (2% of total), but no other races were identified. Polymerase chain reaction and Southern blot analyses with ToxA and ToxB-specific primers and probes were conducted to confirm the race designations of the tested isolates. The ToxA gene was identified in each isolate classified as race 1 or race 2, but not in the race 3 isolate. Sequencing of ToxA from 16 isolates collected in different parts of the province revealed that the sequences were identical. The ToxB gene was not found in isolates classified as races 1 or 2, but a ToxB homologue was identified in the race 3 isolate. These results confirm that the P. tritici-repentis populations in Alberta follow the toxin or inverse gene-for-gene model, and serve to validate the race-based classification system for this pathogen.
... This toxin is found not only in Ptr; it also has been described in other Pyrenophora species such as Pyrenophora bromi (Died.) Drechsler, which produces a homologous ToxB [98]. The ToxA and ToxB activate host resistance responses, similar to that in plant resistance to biotrophic pathogens. ...
The necrotrophic fungus Pyrenophora tritici-repentis is the causal agent of tan spot of wheat, also known as yellow spot. Tan spot is one of the main foliar diseases of wheat, responsible for significant yield loss worldwide. To improve tan spot management, genetic control has been investigated and resistance in some cultivars improved; however, the complexity of the pathosystem wheat - P. tritici-repentis makes integrated disease management strategies very important. In this chapter, we provide an overview of the current state of knowledge of tan spot, including a basic understanding of characterization, pathogenicity, population biology, the global distribution of races, and the genetics of the wheat - P. tritici-repentis interaction. Furthermore, we describe several strategies that can be employed to control tan spot including, seed sanitation, cultural practices, fungicide and biological controls, as well as complementary alternative measures such as fertilization for efficient disease management in wheat production systems.
... Ptr ToxA causes quicker symptoms than Ptr ToxB, though the defense responses observed have multiple similarities [59]. It was also found that Ptr ToxB has a greater distribution than the common host-selective toxins [60]. Pandelova et al. [59] provided an excellent overview of the biochemical mechanisms and effects of both toxins. ...
The genus Pyrenophora includes two important cereal crop foliar pathogens and a large number of less well-known species, many of which are also grass pathogens. Only a few of these have been examined in terms of secondary metabolite production, yet even these few species have yielded a remarkable array of bioactive metabolites that include compounds produced through each of the major biosynthetic pathways. There is little overlap among species in the compounds identified. Pyrenophora tritici-repentis produces protein toxin effectors that mediate host-specific responses as well as spirocyclic lactams and at least one anthraquinone. Pyrenophora teres produces marasmine amino acid and isoquinoline derivatives involved in pathogenesis on barley as well as nonenolides with antifungal activity, while P. semeniperda produces cytochalasans and sesquiterpenoids implicated in pathogenesis on seeds as well as spirocyclic lactams with phytotoxic and antibacterial activity. Less well-known species have produced some unusual macrocyclic compounds in addition to a diverse array of anthraquinones. For the three best-studied species, in silico genome mining has predicted the existence of biosynthetic pathways for a much larger array of potentially toxic secondary metabolites than has yet been produced in culture. Most compounds identified to date have potentially useful biological activity.
... In addition to PDA and potato carrot agar (PCA) media (Xue et al. 2020 Andrie et al. 2008), sterilized wheat straw on PDA (PDA + W) (Marin-Felix et al. 2019), and sterilized wheat straw on water agar (WA + W) for asexual morphs. Microscopic preparations were observed with an ECLIPSE Ti microscope (Nikon, Japan) or an LCD-Digital NSZ-808 stereoscope (NOVEL). ...
Pyrenophora is not only as a genus of pathogens causing leaf damage but also as common seedborne fungi of Italian ryegrass (Lolium multiflorum Lam.). To elucidate the main seedborne Pyrenophora species, 36 seed samples of Italian ryegrass were collected; in total, 113 strains, representing 12.3% of all isolated strains (921), were identified as Pyrenophora species by the identity of ITS sequences in NCBI as well as the similarity of morphological characteristics, of which P. dictyoides (97) was the most frequent species. By pure culture technique, 24 representative pure isolates were eventually retained for further study. Based on DNA analysis of multiple loci (ITS, LSU, GPDH, CHS-1 and RPB1) and morphological characters, eight Pyrenophora species were finally identified, including P. avenicola, P. chaetomioides, P. dictyoides, P. lolii, P. nobleae, P. teres, P. triseptata and P. tritici-repentis; among them, P. avenicola, P. tritici-repentis and P. triseptata were newly reported on Italian ryegrass worldwide. Seed inoculation showed that P. dictyoides, P. lolii and P. teres remarkably decreased the final germination percentages (FGP) and germination indexes (GI) compared with control treatments (P ≤ 0.05); and plant inoculation showed that P. dictyoides, P. lolii and P. nobleae could cause typical brown spot in vivo with higher infection rate (P ≤ 0.05). In conclusion, pathogenicity tests showed that all Pyrenophora species could both inhibit seed germination and infect Italian ryegrass plant to different degrees; among them, P. dictyoides was the most important seedborne pathogen coupled with the isolation and infection rate, followed by P. lolii and P. nobleae. The data generated in this study are helpful for the accurate identification of Pyrenophora species, and the development of seedborne disease management strategies.
... MAT 1-2 yüksek-mobilite grubu (HMG) kutusu, gliseraldehit fosfat dehidrogenaz (GAPDH) ve Internal Transcribed Spacer (ITS) bölgelerinin dizi verilerinin çok genli analizlerine göre Pyrenophora tek bir grupta tanımlanır ve bu analizler tanımlamanın güvenilir olmasını sağlar [26]. ...
Pyrenophora tritici-repentis, (eşeysiz dönemi: Drechslera tritici-repentis) tüm dünyada buğdayda sarı yaprak lekesi hastalığına neden olan önemli bir fungal patojendir. Pyrenophora tritici-repentis Ascomycota funguslarının en büyük ve çeşitli sınıfı olan Dothideomycetes sınıfında yer alır. Fungus, konidiler ile eşeysiz, askosporlar ile eşeyli çoğalabilir. Pyrenophora tritici-repentis karmaşık bir ırk yapısına sahiptir. Irk ayrımı etmen tarafından üretilen üç adet (ToxA, ToxB, ToxC) artık Nekrotrofik Efektör (NE) olarak isimlendirilen konukçuya spesifik toksinlerin (KST) üretimine bağlı olarak yapılmıştır. Efektör üreten her izolatın ırkı ürettiği toksin veya toksinlerin kombinasyonuna göre ayrılır ve toksin üretmiyorsa avirülent ırk olarak kabul edilir. Üretilen bu toksinler etmenin neden olacağı belirtiyi tayin eder. Etmenin hastalık yapabilmesi için konukçu bitkinin söz konusu ırk tarafından üretilen NE'lere karşı hassas olması gerekir. Irk ayrımı ayırıcı set ile yapılır ve bu ayırıcı sete göre etmenin sekiz ırkı tanımlanmıştır. Ayırıcı set altı adet buğday hattı ve çeşidinden oluşur ancak sadece Glenlea çeşidi ve 6B365 ile 6B662 hatları mevcut ırkları etkili biçimde ayırabilmektedir. Ülkemizde de mevcut olan bu hastalık yaprakları etkilediği gibi başak ve taneleri de etkilemektedir. Belirtiler nekroz, kloroz ya da her ikisi olabilir. Tüm belirti durumlarında ilk olarak kahverengi benekler oluşur. Nekrozda kahverengi benekler uzar ve genişler, mercek ya da elmas dilimi şeklinde lezyonlar oluşur. Bu lezyonlar sarı haleler ile sınırlandırılır. Klorozda ise yaprak ayasındaki lezyonları hızla çevreleyen ve gelişen sarı alanlar oluşur. Hastalık, hassas çeşitlerde verimi %50 oranında düşürebilir. Patojen ile etkili biçimde mücadele edebilmek için çeşitli mücadele yöntemlerinin birlikte kullanıldığı entegre mücadele uygulanmalıdır. Bu derlemede buğdayda sarı leke hastalığı ve mücadelesi hakkında bilgi verilmiştir.
... Gene evolution is a process that involves mechanisms such as gene duplications and horizontal gene transfers, which resulted in the hypothesis that sequence-unrelated genes may have high similarity in their tertiary folding and furthermore have the same function in pathogen virulence (Andrie et al., 2008;de Guillen et al., 2015). ...
The wide host range of Xylella fastidiosa (Xf) indicates the existence of yet uncharacterized virulence mechanisms that help pathogen to overcome host-defenses. The availability of various bioinformatics tools and the ability for function prediction of putative virulence proteins, represent a valuable approach for understanding microbial pathogenicity.
Hereby we collected a number of putative effectors from three Xf strains belonging to different subspecies: Temecula-1 (subsp. fastidiosa), CoDiRo (subsp. pauca) and Ann-1 (subsp. sandyi). We designed an in-planta Agrobacterium-based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defense in Nicotiana spp. model plants. Multiple Xf proteins differentially elicited cell death-like phenotypes in different Nicotiana species. These proteins are members of different enzymatic groups: a) hydrolases/hydrolases inhibitors, b) serine proteases and c) metal transferases. We also organized the Xf proteins according to their sequential and structural similarities via the I-TASSER online tool. Interestingly, we identified similar proteins that were able to differentially elicit cell death in different cultivars of the same species.
Our findings provide the bases for further studies on the mechanisms that underlie both defense activation in Xf resistant hosts, as well as pathogens adaptation in susceptible hosts.
... Similarity of NMR structures containing two anti-parallel three-stranded beta sheets with a single disulfide bond has been demonstrated between M. oryzae AVR-Pia, AVR1-CO39, AvrPiz-t and Pyrenophora tritici-repentis ToxB [23,44]. Sequence alignment, position-specific score matrix (PSSM) and profile-HMM searches against these structural homologues had subsequently revealed numerous homologues in other species, including P. bromi [45], Bipolaris oryzae, Colletotrichum spp., Zymoseptoria tritici, Leptosphaeria maculans and even low but significant similarity a protein in plant-associated bacteria Pseudomonas sp. StFLB209 [23]. ...
Plant diseases caused by fungal pathogens are typically initiated by molecular interactions between ‘effector’ molecules released by a pathogen and receptor molecules on or within the plant host cell. In many cases these effector-receptor interactions directly determine host resistance or susceptibility. The search for fungal effector proteins is a developing area in fungal-plant pathology, with more than 165 distinct confirmed fungal effector proteins in the public domain. For a small number of these, novel effectors can be rapidly discovered across multiple fungal species through the identification of known effector homologues. However, many have no detectable homology by standard sequence-based search methods. This study employs a novel comparison method (RemEff) that is capable of identifying protein families with greater sensitivity than traditional homology-inference methods, leveraging a growing pool of confirmed fungal effector data to enable the prediction of novel fungal effector candidates by protein family association. Resources relating to the RemEff method and data used in this study are available from https://figshare.com/projects/Effector_protein_remote_homology/87965 .
... Other markers that were significantly associated with genetic structuring of the P. teres f. teres populations included a glyceraldehyde-3-phosphate dehydrogenase-like protein (GPD1) gene, an endo-1,4-betaxylanase A mRNA gene, and a cytochrome P450 lanosterol 14 alpha-demethylase (CYP51A) gene. The GPD1 gene has been frequently used as a genetic marker in phylogenetic studies to differentiate fungal pathogens including P. teres (Andrie et al. 2008;Lu et al. 2013;Zhang and Berbee 2001). GPD1 plays a major role in fungal metabolic pathways such as energy synthesis and biomass synthesis (Larsson et al. 1998). ...
Net-form net blotch disease caused by Pyrenophora teres f. teres (Ptt) results in significant yield losses to barley industries. Up-to-date knowledge of the genetic diversity and structure of pathogen populations is critical for better understanding the disease epidemiology and unravelling pathogen survival and dispersal mechanisms. Thus, this study investigated long distance dispersal and adaptation by analysing the genetic structure of 250 Ptt isolates collected from Australia, Canada, Hungary and Republic of South Africa (RSA), and historical isolates from Canada, Denmark, Japan and Sweden. The population genetic structure detected by discriminant analysis of principal component, using 5890 Diversity Arrays Technology (DArT) markers, revealed the presence of four clusters. Two of these contained isolates from all regions, and all isolates from RSA were grouped in these two. Australia and Hungary showed three clusters each. One of the Australian clusters contained only Australian isolates. One of the Hungarian clusters contained only Hungarian isolates and one Danish isolate. STRUCTURE analysis indicated that some isolates from Australia and Hungary shared recent ancestry with RSA, Canada and historical isolates and were thus admixed. Subdivisions of the Neighbor-joining network indicated that isolates from distinct countries were closely related, suggesting multiple introduction events conferred genetic heterogeneity in these countries. Through a Neighbor-joining analysis and amplification with form-specific DNA markers two hybrid isolates, CBS 281.31 from Japan and H-919 from Hungary collected in 1931 and 2018, respectively, were detected. These results provide a foundation for exploring improved management of disease incursions and pathogen control through strategic deployment of resistances.
... These results, together with ours, provide evidence that there is possibly a common genomic region on chromosome 1A associated with resistance to race 1 isolates, irrespective of the type of toxins produced by the fungus (Ptr ToxA, Ptr ToxC, or both). Besides Ptr ToxA and ToxC, other studies have also suggested the role of other genes or QTLs on the virulence of race 1 isolates (Effertz et al., 2002;Friesen et al., 2002;Andrie et al., 2008;Chu et al., 2008b). ...
Leaf rust, yellow (stripe) rust, common bunt, and tan spot are economically significant diseases affecting wheat (Triticum aestivum L.) production in Canada. In this study, we investigated the genetic relationship and population structure of 81 Canadian western spring wheat cultivars released primarily between 1963 and 2011 and identified genomic regions associated with resistance to the above four diseases and insensitivity to three Pyrenophora tritici‐repentis (Ptr) toxins (Ptr ToxA, Ptr ToxB and Ptr ToxC). The cultivars were evaluated for field reaction to the four diseases and for reaction to the three Ptr toxins in a greenhouse and were genotyped with a subset of 19,919 of the wheat 90K single‐nucleotide polymorphic array and 11 gene‐specific markers. There were large genetic differences among pairwise comparisons of cultivars, except six pairs that showed <0.05 genetic distance. The cultivars exhibited clear population structure, generally in agreement with the major western Canada spring wheat classes. Using a threshold of p ≤ 5 × 10−5 and a weighted mixed linear model, we identified 94 markers from seven chromosomes associated with all traits except Ptr ToxC. Two major‐effect genomic regions on chromosomes 5B (71–74 cM) and 1A (52–53 cM) were associated with Ptr ToxA, of which the former coincided with the Tsn1 gene. For Ptr ToxB, we identified two other major‐effect regions on chromosomes 2B and 5B. The genomic regions associated with common bunt mapped on 2B, 4B, and 7A, whereas those associated with leaf rust mapped at two positions on 2B. We were only able to uncover a single marker‐trait association for tan spot on 7B and for yellow rust on 2A.
... MAX effectors represent the sequence-unrelated, structurally similar effectors in M. oryzae. Their origin was, however, suggested to be ancient, and duplications as well as diversifying evolution more likely explain the frequent occurrence of these effectors within an organism and across the phytopathogens (16,74). With protein structure prediction, we also illustrated that putative effectors with the alpha-beta plait topology or the γ -crystallin-like fold may be sequence-unrelated but structurally similar. ...
Magnaporthe oryzae relies on a diverse collection of secreted effector proteins to reprogram the host metabolic and immune responses for the pathogen's benefit. Characterization of the effectors is thus critical for understanding the biology and host infection mechanisms of this phytopathogen. In rapid, divergent effector evolution, structural information has the potential to illuminate the unknown aspects of effectors that sequence analyses alone cannot reveal. It has recently become feasible to reliably predict the protein structures without depending on homologous templates. In this study, we tested structure modeling on 1854 secreted proteins from M. oryzae and evaluated success and obstacles involved in effector structure prediction. With sensitive homology search and structure-based clustering, we defined both distantly related homologous groups and structurally related analogous groups. With this dataset, we propose sequence-unrelated, structurally similar effectors are a common theme in M. oryzae and possibly in other phytopathogens. We incorporated the predicted models for structure-based annotations, molecular docking and evolutionary analyses to demonstrate how the predicted structures can deepen our understanding of effector biology. We also provide new experimentally testable structure-derived hypotheses of effector functions. Collectively, we propose that computational structural genomic approaches can now be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of reliable, machine learning-based structure prediction.
... ToxB-like sequences are also found in race 3 isolates of Ptr, other species of the genus Pyrenophora, and even other genera of the Pleosporacea (Martinez et al., 2004;Strelkov et al., 2006;Andrie et al., 2007). Isolates of Pyrenophora bromi, a sister species to Ptr causing brown spot of brome grass, has several ToxB-like sequences (termed Pb ToxB) with coding regions having 89% similarity to ToxB (Andrie et al., 2008;Andrie and Ciuffetti, 2011). However, none of the heterologously expressed Pb ToxB proteins induced symptoms on brome grass, while they did cause chlorosis on ToxB-sensitive wheat genotypes (Andrie and Ciuffetti, 2011). ...
The fungus Pyrenophora tritici-repentis (Ptr) causes tan spot, a destructive foliar disease of wheat worldwide. The pathogen produces several necrotrophic effectors, which induce necrosis or chlorosis on susceptible wheat lines. Multiple races of Ptr have been identified, based on their ability to produce one or more of these effectors. Ptr has a wide host range of cereal and non-cereal grasses, but is known to cause damage only on wheat. Previously, we showed that Ptr can interact specifically with cultivated barley (Hordeum vulgare ssp. vulgare), and that the necrotrophic effector Ptr ToxB induces mild chlorosis in a highly selective manner when infiltrated into certain barley genotypes. In the present study, a barley doubled-haploid (DH) population was evaluated for reaction to Ptr race 5, a Ptr ToxB-producer. Then a comprehensive genetic map composed of 381 single nucleotide polymorphism (SNP) markers was used to map the locus conditioning this chlorosis. The F1 seedlings, and 92 DH lines derived from a cross between the resistant Japanese malting barley cultivar Haruna Nijo and the susceptible wild barley (H. vulgare ssp. spontaneum) OUH602 were inoculated with a conidial suspension of Ptr race 5 isolate at the two-leaf stage. The seedlings were monitored daily for symptoms and assessed for chlorosis development on the second leaf, 6 days after inoculation. All tested F1 seedlings exhibited chlorosis symptoms similar to the susceptible parent, and the DH lines segregated 1:1 for susceptible:resistant phenotypes, indicating the involvement of a single locus. Marker-trait linkage analysis based on interval mapping identified a single locus on the distal region of the short arm of chromosome 2H. We designate this locus Susceptibility to P. tritici-repentis1 (Spr1). The region encompassing this locus has 99 high confidence gene models, including membrane receptor-like kinases (RLKs), intracellular nucleotide-binding, leucine-rich repeat receptors (NLRs), and ankyrin-repeat proteins (ANKs). This shows the involvement of a dominant locus conferring susceptibility to Ptr in barley. Further work using high-resolution mapping and transgenic complementation will be required to identify the underlying gene.
... Yet in the same Ptr species, there are no homologs of this gene in isolates lacking the ability to produce the Ptr ToxA effector, and only the producing isolates carry the single copy ToxA gene (Aboukhaddour et al., 2009). On the other hand, homologs of ToxB were found in other fungal species of Cochliobolus, Alteranaria, and Pyrenophora (Andrie et al., 2008;Andrie and Ciuffetti, 2011), and in the Ptr isolates that do not produce the active Ptr ToxB protein. ...
Tan spot is a destructive foliar wheat disease worldwide and caused by the ascomycete fungus Pyrenophora tritici–repentis (Ptr); it has become more frequent in Tunisia over the last decade. In this study, the virulence of 73 single-spore isolates, collected from durum and bread wheat fields during 2017–2018 growing season, was evaluated on four differential wheat genotypes. This was followed by polymerase chain reaction tests with specific primers for the effector genes ToxA, ToxB, and toxb (ToxB-homolog). Sequence analysis to validate the identity of the amplified genes was followed, and ToxA amplicons from a subset of 22 isolates were analyzed to determine its haplotype identity. Ptr isolates from Tunisia were grouped in races 2, 4, 5, and 7, and 44% of the tested isolates did not fit under any known race, and were denoted here as atypical. These atypical isolates induced the same symptoms as race 7 isolates, extensive necrosis, and chlorosis on susceptible genotypes, but lacked the ToxA gene. ToxA is the only identified necrosis-inducing effector in Ptr, and was amplified in 51% of tested isolates, and shared identical sequence to previously identified haplotype (H15). ToxB and its homolog toxb were present in 97% and 93% of tested isolates, respectively. Ptr in Tunisia lacked Ptr ToxC activity, and none of the tested isolates induced the specific symptoms of that effector. Race 7 and the atypical isolates dominated the Tunisian Ptr population, while races 2, 4, and 5 were found at low percentages. In conclusion, ToxB and its homolog were the most dominant genes in Ptr from Tunisia, and the majority of the isolates induced necrosis and chlorosis on Ptr ToxA and Ptr ToxB susceptible wheat genotypes. However, only about half of that necrosis can be attributed to ToxA presence, this result necessitates further research to investigate the prevalence of additional necrotic effector(s).
Terminology: in this paper, Pyrenophora tritici–repentis abbreviated as Ptr, the effectors are referred to by Ptr ToxA, Ptr ToxB and Ptr ToxC, and the genes coding for them are written in italic as ToxA, ToxB, and ToxC, respectively.
... We have also tested the potential use of ITS sequences to construct a phylogenetic tree. Although ITS serves as a good "barcode" for differentiating fungal species [14], it did not contain sufficient variability to generate genetic resolution between isolates of P. teres (i.e., isolates of the same specie), as previously demonstrated by Andrie et al. [27]. ...
Net blotch (NB) is a major disease of barley caused by the fungus Pyrenophora teres f. teres (Ptt), and P. teres f. maculata (Ptm). Ptt and Ptm infect the cultivated crop (Hordeum vulgare) and its wild relatives (H. vulgare ssp. spontaneum and H. murinum ssp. glaucum). The main goal of this research was to study the NB-causing pathogen in the crop center of origin. To address this, we have constructed a Ptt (n = 15) and Ptm (n = 12) collection isolated from three barley species across Israel. Isolates were characterized genetically and phenotypically. Aggressiveness of the isolates was determined based on necrotrophic growth rate on detached leaves of barley. In addition, isolates were genetically characterized by the mating type, followed by phylogenetic analysis, clustering them into seven groups. The analysis showed no significant differentiation of isolates based on either geographic origin, host of origin or form (Ptt vs. Ptm). Nevertheless, there was a significant difference in aggressiveness among the isolates regardless of host species, geographic location or sampling site. Moreover, it was apparent that the isolates derived from wild hosts were more variable in their necrotrophic growth rate, compared to isolates sampled from cultivated hosts, thereby suggesting that NB plays a major role in epidemiology at the center of barley origin where most of the diversity lies. Ptm has significantly higher necrotrophic and saprotrophic growth rates than Ptt, and for both a significant negative correlation was found between light intensity exposure and growth rates.
... These results, together with ours, provide evidence that there is possibly a common genomic region on chromosome 1A associated with resistance to race 1 isolates, irrespective of the type of toxins produced by the fungus (Ptr ToxA, Ptr ToxC, or both). Besides Ptr ToxA and ToxC, other studies have also suggested the role of other genes or QTLs on the virulence of race 1 isolates (Effertz et al., 2002;Friesen et al., 2002;Andrie et al., 2008;Chu et al., 2008b). ...
Leaf rust, yellow (stripe) rust, common bunt, and tan spot are economically significant diseases affecting wheat (Triticum aestivum L.) production in Canada. In this study, we investigated the genetic relationship and population structure of 81 Canadian western spring wheat cultivars released primarily between 1963 and 2011 and identified genomic regions associated with resistance to the above four diseases and insensitivity to three Pyrenophora tritici-repentis (Ptr) toxins (Ptr ToxA, Ptr ToxB and Ptr ToxC). The cultivars were evaluated for field reaction to the four diseases and for reaction to the three Ptr toxins in a greenhouse and were genotyped with a subset of 19,919 of the wheat 90K single-nucleotide polymorphic array and 11 gene-specific markers. There were large genetic differences among pairwise comparisons of cultivars, except six pairs that showed <0.05 genetic distance. The cultivars exhibited clear population structure, generally in agreement with the major western Canada spring wheat classes. Using a threshold of p ≤ 5 × 10⁻⁵ and a weighted mixed linear model, we identified 94 markers from seven chromosomes associated with all traits except Ptr ToxC. Two major-effect genomic regions on chromosomes 5B (71–74 cM) and 1A (52–53 cM) were associated with Ptr ToxA, of which the former coincided with the Tsn1 gene. For Ptr ToxB, we identified two other major-effect regions on chromosomes 2B and 5B. The genomic regions associated with common bunt mapped on 2B, 4B, and 7A, whereas those associated with leaf rust mapped at two positions on 2B. We were only able to uncover a single marker-trait association for tan spot on 7B and for yellow rust on 2A.
... Homologues of ToxB (toxb) are also found in isolates of Ptr that lack Ptr ToxB activity (Martinez et al., 2004;Strelkov et al., 2006). Additionally, ToxB-like sequences have been reported from Pyrenophora bromi and other members of the family Pleosporaceae (Andrie et al., 2008). Heterologously expressed 'Pb ToxB' from P. bromi, which causes brown spot of brome grass, induced chlorosis in Ptr ToxBsensitive genotypes of wheat (Andrie & Ciuffetti, 2011). ...
... Homologues of ToxB (toxb) are also found in isolates of Ptr that lack Ptr ToxB activity (Martinez et al., 2004;Strelkov et al., 2006). Additionally, ToxB-like sequences have been reported from Pyrenophora bromi and other members of the family Pleosporaceae (Andrie et al., 2008). Heterologously expressed 'Pb ToxB' from P. bromi, which causes brown spot of brome grass, induced chlorosis in Ptr ToxBsensitive genotypes of wheat (Andrie & Ciuffetti, 2011). ...
Pyrenophora tritici-repentis (Ptr) is a destructive fungal pathogen of wheat worldwide. In addition to wheat, Ptr has been isolated from various other hosts in the family Poaceae, yet the nature of its interaction with those hosts is unknown. The Ptr-barley relationship was explored and the existence of a specific interaction between Ptr and barley is described for the first time; symptom development on several barley genotypes was evaluated in bioassays and by toxin infiltration into barley leaves. Ptr ToxB-producing isolates of the fungus were able to cause significant damage when inoculated onto certain barley genotypes, and Ptr ToxB was able to induce chlorosis in a highly selective manner when infiltrated into those same genotypes. Ptr-barley specificity is subtle and can break with slight changes in temperature after infection. To understand the infection process in barley, a cytological analysis and in planta fungal biomass estimation using quantitative PCR were performed. The fungus penetrates through the host epidermal cells and advances to colonize the mesophyll layer intercellularly, with the infection process on barley closely resembling that on wheat. Here, evidence is provided for a specific interaction between barley and Ptr, expanding understanding of Ptr-host specificity and breaking the assumption that the highest level of specificity seen with Ptr is restricted to particular genotypes of the wheat host.
... Genome sequencing identified a homolog of ToxA gene in S. nodorum that has been interpreted as support for horizontal transfer of ToxA to P. tritici-repentis [42]. By contrast, the ToxB gene is widely distributed across ascomycete phylogeny [43]. Although most HSTs have restricted distributions, phylogenomic analyses have identified homologs of core genes in other Pezizomycotina, but their function as HSTs is only known for Dothideomycetes [36 ,44,45]. ...
... The multicopy nature of ToxB, the existence of toxb in P. tritici-repentis, and the presence of ToxB homologs in other ascomycetes (Andrie and Ciuffetti 2011;Andrie et al. 2008;Martinez et al. 2001Martinez et al. , 2004Strelkov et al. 1999Strelkov et al. , 2006 suggest that ToxB could have functions other than that of a chlorosisinducing toxin (Amaike et al. 2008;Ciuffetti et al. 2010). Interestingly, silencing of ToxB in P. tritici-repentis reduces the ability to induce chlorosis and the number of appressoria formed compared with wild-type strains (Aboukhaddour et al. 2012). ...
The necrotrophic fungus Pyrenophora tritici-repentis (Ptr) is responsible for the disease tan spot of wheat. Ptr ToxB (ToxB), a proteinaceous host-selective toxin, is one of the effectors secreted by Ptr. ToxB induces chlorosis in toxin sensitive wheat cultivars and displays characteristics common to apoplastic effectors. We addressed the hypothesis that ToxB exerts its activity extracellularly. Our data indicate that hydraulic pressure applied in the apoplast following ToxB infiltration can displace ToxB-induced symptoms. In addition, treatment with a proteolytic cocktail following toxin infiltration results in reduction of symptom development and indicates that ToxB requires at least 8 hours in plantato induce maximum symptom development. In vitro assays demonstrate that apoplastic fluids extracted from toxin sensitive and insensitive wheat cultivars cannot degrade ToxB. Additionally, ToxB can be re-isolated from apoplastic fluid after toxin infiltration. Furthermore, localization studies of fluorescently-labeled ToxB indicate that the toxin remains in the apoplast in toxin sensitive and insensitive wheat cultivars. Our findings support the hypothesis that ToxB acts as an extracellular effector.
... Thus, Ptr ToxC may be the major pathoginicity factor in many susceptible accessions. In addition, we may not be able to rule out other factors as virulence determinants of race 1 of P. tritici-repentis (Friesen et al., 2002;Andrie et al., 2007;Eff ertz et al., 2002) besides Ptr ToxA and Ptr ToxC. Chu et al. (2008b) studied tan spot resistance in T. dicoccoides and suggested that other genes besides Tsn1/tsn1 might be involved in governing susceptibility or resistance to tan spot. ...
Tan spot, caused by the fungus Pyrenophora tritici‐repentis, is an important foliar disease of wheat (Triticum aestivum L.) worldwide. Growing resistant cultivars is an effective approach to reduce the losses caused by the disease. To identify resistance genes in common wheat, 380 wheat accessions from different geographical origins were evaluated for resistance to P. tritici‐repentis race 1, the predominant race in the Great Plains of the United States and western Canada, and insensitivity to Ptr ToxA, a host‐selective toxin produced by race 1. Most accessions tested (60%) were resistant and only 93 accessions (24%) were as susceptible as TAM 105, the susceptible control. Among 379 accessions, 230 were insensitive to Ptr ToxA, but only 158 of them showed resistance to race 1. A weak correlation between tan spot score and sensitivity to Ptr ToxA suggests that pathogenicity factors other than Ptr ToxA (like Ptr ToxC) also contributed to tan spot development in these accessions. The accessions with resistance to tan spot identified in this study should be useful sources for developing new tan spot resistant cultivars.
... Wheat sensitivity is conditioned by the dominant Tsc2 gene, which has been mapped to the short arm of chromosome 2B (Strelkov et al. 1999;Friesen and Faris 2004). Unlike most effectors, PtrToxB homologues have been found across a broad range of plant pathogenic ascomycetes, suggesting that it may have arisen in an early ancestor of the Ascomycota (Andrie et al. 2008). However, whether PtrToxB and its homologues play a role in plant-microbe interactions is yet to be elucidated. ...
The host-pathogen interface can be considered as a biological battlefront. Molecules produced by both the pathogen and the host are critical factors determining the outcome of the interaction. Recent studies have revealed that an increasing number of necrotrophic fungal pathogens produce small proteinaceous effectors that are able to function as virulence factors. These molecules can cause tissue death in host plants that possess dominant sensitivity genes, leading to subsequent pathogen colonisation. Such effectors are only found in necrotrophic fungi, yet their roles in virulence are poorly understood. However, several recent key studies of necrotrophic effectors from two wheat (Triticum aestivum L.) pathogens, Pyrenophora tritici-repentis (Died.) Drechs. and Stagonospora nodorum (Berk.) Castell. & Germano, have shed light upon how these effector proteins serve to disable the host from the inside out.
... Thus, Ptr ToxC may be the major pathoginicity factor in many susceptible accessions. In addition, we may not be able to rule out other factors as virulence determinants of race 1 of P. tritici-repentis (Friesen et al., 2002;Andrie et al., 2007;Eff ertz et al., 2002) besides Ptr ToxA and Ptr ToxC. Chu et al. (2008b) studied tan spot resistance in T. dicoccoides and suggested that other genes besides Tsn1/tsn1 might be involved in governing susceptibility or resistance to tan spot. ...
Pyrenophora tritici-repentis is a destructive pathogen of wheat with global impact. It possesses a highly plastic open pangenome shaped by the gain and loss of effector genes. This study investigated the allelic variations in the chlorosis-encoding gene, ToxB, across 422 isolates representing all identified pathotypes and worldwide origins. To gain better insights into ToxB evolution, we examined its presence and variability in other Pyrenophora spp. A ToxB haplotype network was constructed, revealing the evolutionary relationships of this gene (20 haplotypes) across four Pyrenophora species. Notably, toxb, the homolog of ToxB, was detected for the first time in the barley pathogen Pyrenophora teres. The ToxB/toxb genes display evidence of selection that is characterized by loss of function, duplication, and diverse mutations. Among ToxB/toxb open reading frame, 72 mutations were identified, including 14 synonymous, 55 nonsynonymous, and 3 indel mutations. Remarkably, a ~5.6 Kb Copia-like retrotransposon, named Copia-1_Ptr, was found inserted in the toxb gene of a race 3 isolate. This insert disrupted the ToxB gene's function, a first case of effector gene disruption by a transposable element in Ptr. Additionally, a microsatellite with 25-nucleotide repeats (0 to 10) in the upstream region of ToxB suggested a potential mechanism influencing ToxB expression and regulation. Exploring ToxB-like protein distribution in other Ascomycetes revealed their presence in 19 additional species, including the Leotiomycetes class for the first time. The presence/absence pattern of ToxB-like proteins defied species relatedness compared to a phylogenetic tree, suggesting a past horizontal gene transfer event.
ToxA is one of the most studied proteinaceous necrotrophic effectors produced by plant pathogens. It has been identified in four pathogens (Pyrenophora tritici-repentis, Parastagonospora nodorum, Parastagonospora pseudonodorum (formerly Parastagonospora avenaria f. sp. tritici) and Bipolaris sorokiniana) causing leaf spot diseases on cereals worldwide. To date, 24 different ToxA haplotypes have been identified. Some Py. tritici-repentis and related species also express ToxB, another small protein necrotrophic effector. We present here a revised and standardized nomenclature for these effectors, which could be extended to other poly-haplotypic (allelic) genes found across multiple species.
Alternaria leaf spot, caused by Alternaria spp., is a common foliar disease of sugar beet and other Beta vulgaris crop types. Known to be present in the US since at least the 1950s, this disease has been found in all major sugar beet growing regions of the US as well being reported as present everywhere beets are grown. Alternaria leaf spot symptoms are similar to several other common foliar diseases of beets including Cercospora leaf spot and the early stages of Phoma leaf spot. Symptoms and signs of this disease in the field do not provide enough information to help distinguish between diseases unless the viewer is readily familiar with the pathogen, especially when viewed with the unaided eye. Alternaria cultures can be identified to species using colony and conidial morphology on select culture media or using molecular markers. While not as common as Cercospora leaf spot, Alternaria leaf spot can result in significant reductions in yield and quality when not managed.
Taproot decline (TRD) of soybean [Glycine max (L) Merr.] is an emerging disease caused by Xylaria necrophora, and currently observed in the southern United States. X. necrophora infects soybean roots, causing necrosis and foliar interveinal chlorosis followed by necrosis. The most recent estimates of yield losses associated with this disease (0.57 - 1.18 million bushels) highlight its potential to become a major problem for producers in the region. The mechanism by which X. necrophora affects soybean remains unknown, but a plausible explanation is that phytotoxic secondary metabolites (SMs) are produced by X. necrophora in the roots. To test this hypothesis, cell-free culture filtrates (CFCFs) from three X. necrophora strains and one putative Colletotrichum siamense were used to challenge soybean stem cuttings to determine if foliar symptoms of TRD are caused by SMs, tolerance to SMs among potentially resistant soybean cultivars identified in greenhouse and field experiments, and specificity of SMs across plant species (cotton, peanut, tomato, and soybean). Measurements of chlorophyll content and root length were obtained at 7 and 14 days of exposure (DOE). Lower chlorophyll content and shorter roots were observed on all soybean cultivars treated with CFCFs of pathogenic isolates and no effects on other plant species were observed, suggesting X. necrophora produces SMs that are detrimental to soybean exclusively and resistance upon direct exposure to SMs does not exist. These results suggest soybean-specific SMs are produced by X. necrophora in the roots causing the interveinal chlorosis and subsequent necrosis observed on plants affected by TRD.
We sequenced the genome of a global collection (40 isolates) of the fungus Pyrenophora tritici-repentis (Ptr), a major foliar pathogen of wheat and model for the evolution of necrotrophic pathogens. Ptr exhibited an open-pangenome, with 43% of genes in the core set and 57% defined as accessory (present in only a subset of isolates), of which 56% were singleton genes (present in only one isolate). A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around the effector coding genes, were explored further using the annotated genomes of two isolates sequenced by PacBio RS II and Illumina HiSeq. The Ptr genome exhibited major chromosomal rearrangements, including chromosomal fusion, translocation, and segment duplications. An intraspecies translocation of ToxA , the necrosis-inducing effector-coding gene, was facilitated within Ptr via a 143 kb ‘ Starship’ transposon (dubbed ‘Horizon’). Additionally, ToxB , the gene encoding the chlorosis-inducing effector, was clustered as three copies on a 294 kb transposable element in a ToxB-producing isolate. ToxB and its carrying transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and the transposon were both present in another non-coding isolate. The Ptr genome also appears to exhibit a ‘one-compartment’ organization, but may still possess a ‘two-speed genome’ that is facilitated by copy-number variation as reported in other fungal pathosystems.
IMPORTANCE
Ptr is one of the most destructive wheat pathogens worldwide. Its genome is a mosaic of present and absent effectors, and serves as a model for examining the evolutionary processes behind the acquisition of virulence in necrotrophs and disease emergence. In this work, we took advantage of a diverse collection of pathogenic Ptr isolates with different global origins and applied short- and long-read sequencing technologies to dissect the Ptr genome. This study provides comprehensive insights into the Ptr genome and highlights its structural organization as an open pangenome with ‘one-compartment’. In addition, we identified the potential involvement of transposable elements in genome expansion and the movement of virulence factors. The ability of effector-coding genes to shuffle across chromosomes on large transposons was illustrated by the intraspecies translocation of ToxA and the multi-copy ToxB . In terms of gene contents, the Ptr genome exhibits a large percentage of orphan genes, particularly in non-pathogenic or weakly-virulent isolates.
Barley (Hordeum vulgare) is generally used for animal feed, and food and beer production industries. Its production is affected by phytopathogens, of which Ramularia collo-cygni, Puccinia spp., Pyrenophora teres and Cochliobolus sativus are of global concern. In Mexico, there are few studies available on the morphological and molecular identification of fungi responsible for causing fungal diseases of barley. Therefore, the objective of this work was to identify, morphologically and molecularly, fungi isolated from barley seeds. The strains were identified by morphological analysis and by sequencing of the conserved ITS1/5.8s/ITS2 region of the ribosomal gene. Morphologically, the strains were identified as P. teres and C. sativus, which was confirmed with bioinformatics techniques using BLAST and MEGA6 programs. The results showed that the strains isolated from barley seeds in the highlands of Mexico were consistent with the sequences of P. teres and C. sativus deposited in the GenBank. These results will allow identifying some of the species of native fungi found in barley seeds.
Structural biology has the potential to illuminate the evolution of pathogen effectors and their commonalities that cannot be readily detected at the primary sequence level. Recent breakthroughs in protein structure modeling have demonstrated the feasibility to predict the protein folds without depending on homologous templates. These advances enabled a genome-wide computational structural biology approach to help understand proteins based on their predicted folds. In this study, we employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, we predicted the folds of 1,295 proteins (70%). We showed that template-free modeling by TrRosetta captured 514 folds missed by homology modeling, including many known effectors and virulence factors, and that TrRosetta generally produced higher quality models for secreted proteins. Along with sensitive homology search, we employed structure-based clustering, defining not only homologous groups with divergent members but also sequence-unrelated structurally analogous groups. We demonstrate that this approach can reveal new putative members of structurally similar MAX effectors and novel analogous effector families present in M. oryzae and possibly in other phytopathogens. We also investigated the evolution of expanded putative ADP-ribose transferases with predicted structures. We suggest that the loss of catalytic activities of the enzymes might have led them to new evolutionary trajectories to be specialized as protein binders. Collectively, we propose that computational structural genomics approaches can be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of machine learning-based structure prediction.
[Formula: see text]Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
A dead woody sample of Acer sp. with fungal fruiting bodies was collected in Pu’er City of Yunnan Province. Multigene phylogenetic analyses of LSU, ITS, SSU, and tef1-α sequence data showed that our collection belongs to Montagnula and is well separated from all other extant species. Montagnula puerensis is compared with all extant species by morphological characteristics, culture characteristics, host, and location information and is the first report of Montagnula from the host genus Acer.
A new leaf blight disease of browntop millet (Brachiaria ramosa) was noticed during rainy season (Kharif) 2018 at small millet experimental field, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra (GKVK), Bengaluru, India. To assess the disease severity, an intensive roving survey was conducted during the 2019 cropping season. Based on the morphological characterization, the causal agent of leaf blight disease was identified as Bipolaris spp. Further sequencing and combined gene analysis of ITS (internal transcribed spacer of rDNA), GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) and LSU (large subunit) of all the nine isolates confirmed the pathogen as B. setariae. Pathogenicity study showed that all the isolates were pathogenic and caused leaf blight symptoms on browntop millet. The B. setariae isolates showed marked variability with respect to disease incidence on browntop millet (cv. Dundu korale) under artificial inoculation conditions. However, the host range was limited only to browntop millet and found non‐pathogenic to other six small millets examined. To our knowledge, this is the first completely described study on characterization of B. setariae causing leaf blight disease of browntop millet in India.
Pyrenophora tritici-repentis is an ascomycete fungus that causes tan spot of wheat. The disease has a worldwide distribution and can cause significant yield and quality losses in wheat production. The fungal pathogen is homothallic in nature, which means it can undergo sexual reproduction by selfing to produce pseudothecia on wheat stubble for seasonal survival. Since homothallism precludes the development of bi-parental fungal populations, no genetic linkage map has been developed for P. tritici-repentis for mapping and map-based cloning of fungal virulence genes. In this work, we created two heterothallic strains by deleting one of the mating type genes in each of two parental isolates 86-124 (race 2) and AR CrossB10 (a new race) and developed a bi-parental fungal population between them. The draft genome sequences of the two parental isolates were aligned to the Pt-1C-BFP reference sequence to mine single nucleotide polymorphisms (SNPs). A total of 225 SNP markers were developed for genotyping the entire population. Additionally, 75 simple sequence repeat, and two gene markers were also developed and used in the genotyping. The resulting linkage map consisted of 13 linkage groups spanning 5,075.83 cM in genetic distance. Because the parental isolate AR CrossB10 is a new race and produces Ptr ToxC, it was sequenced using long-read sequencing platforms and de novo assembled into contigs. The majority of the contigs were further anchored into chromosomes with the aid of the linkage maps. The whole genome comparison of AR CrossB10 to the reference genome of M4 revealed a few chromosomal rearrangements. The genetic linkage map and the new AR CrossB10 genome sequence are valuable tools for gene cloning in P. tritici-repentis.
The dothideomycete genera Bipolaris and Curvularia are important pathogens of grasses (Poaceae) and can associate with host tissues without disease symptoms. Species of both genera are responsible for leaf spot epidemics that commonly occur on turfgrass throughout the United States transition zone and southern regions. Recent species typifications with DNA sequences have made a broad examination of Bipolaris and Curvularia species obtained from turfgrass possible. Isolates for analysis were obtained from culture collections or isolated from symptomatic turfgrass samples. Phylogenies based on ITS, GPD, and TEF gene sequences were constructed using maximum likelihood and Bayesian analyses and used to determine intraspecific and intrageneric relationships among Bipolaris and Curvularia species isolated from turfgrasses. Similar to previous studies, Bipolaris and Curvularia species formed separate, well‐supported clades. Morphological identifications of genera and species based on published host‐based keys often disagreed with molecular groupings. For example, C. hawaiiensis (= B. hawaiiensis) could not be differentiated from B. cynodontis while C. australiensis (= B. australiensis) could not be differentiated from C. spicifera (= B. spicifera). Additionally, phylogenies suggest B. coffeana and B. cynodontis are synonymous. However, the number of pseudosepta per conidium was the only consistent morphological character. The delineation of species causing leaf spot diseases of warm‐season turfgrasses should improve disease management decisions and help identify potential pathogens in asymptomatic tissues.
In wheat, genes for resistance (R) as well as susceptibility (S) are now known for several diseases. The S genes also include sensitivity genes like Tsn1 in wheat. R genes follow a gene-for-gene (GFG) relationship and generally involve biotrophs and S genes particularly sensitivity genes, follow an inverse gene-for-gene relationship (IGFG), generally involving necrotroph or hemi-biotroph pathogens. The toxin (virulence factor) genes of the pathogen and the corresponding sensitivity genes have been described in some detail for the following three pathogens: (i) Paratagonospora nodorum (causing Septoria nodorum blotch or SNB); (ii) Pyrenophora tritici - repentis (tan spot) and (iii) Bipolaris sorokiniana (spot blotch). These and some other pathogens produce several necrotrophic effectors (NEs), which interact directly or indirectly with the products of S genes in the host and produce disease symptoms like necrosis and/or chlorosis. In this article we present a critical review of all the relevant information about the interactions between NEs of the above three pathogens and the corresponding S genes in wheat. The gaps in knowledge and possibilities for future research are also discussed.
Pyrenophora teres f. teres is well known for causing net form net blotch on barley, but its pathogenicity on wheat has also been reported in Hungary (Tóth et al. 2008) and east Russia (Mikhailova et al. 2010). The report from Hungary mentioned symptoms of dark brown necrotic lesions with or without chlorotic margins, while the report from Russia described symptoms similar to tan spot. A survey of fungal pathogens causing leaf spot diseases on wheat in the humid subtropical areas of Paraná State, southern Brazil, was conducted in 2016. Twenty four isolates, representing different wheat fields in the municipalities of Guarapuava and Pinhão, in the state of Paraná, were collected from wheat plants at the soft dough stage. The isolates analyzed in our study were obtained from leaf fragments of wheat cv. Toruk and were removed from the edge of lesions resembling tan spot, a major fungal disease of wheat in that region, caused by Pyrenophora tritici-repentis. The leaves presented oval, diamond, or elongate-shape lesions with tan necrotic spots. Still, they did not always show the brown center, which seemed not very characteristics of tan spot caused by P. tritici-repentis. Yellow halos surrounded the necrotic lesions. The fragments were superficially disinfected for 30 sec in 0.5% sodium hypochlorite, washed in sterile water, dried with a sterile paper towel, and transferred to petri plates containing potato dextrose agar (PDA) medium. After four days of incubation at 24±2ºC and a photoperiod of 12 h, hyphal tips were removed from the PDA medium and transferred to petri plates containing V-8 medium. Plates were incubated for 14 days in alternate cycles of 12 h at 23±2ºC under light and 12 h at 19±2ºC in darkness for sporulation. Pure cultures were obtained from single conidia produced on V-8 medium. Single spore isolates, representing different fields, were obtained from individual leaves. Conidia measuring 38–82 µm × 16–20 µm were smoothly cylindrical and straight, with rounded ends, subhyaline or yellowish brown, with four to seven pseudosepta. Genomic DNA extracted from cultures grown in yeast extract broth medium was used for PCR amplification of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene (ITS-5.8S rDNA) using the primers ITS4 and ITS5 described by White et al. (1990). A BLAST search of the GenBank database revealed that the ITS-5.8S rDNA sequence fragments (505 bp) from 12 out of 67 isolates were 99-100% similar to P. teres accession number EF452452 (Andrie et al. 2008). The DNA sequences of three P. teres isolates (20C3, 20A3, and 20A1) were deposited in GenBank (accession nos. MH594043.1, MH594042.1, and MH594041.1, respectively). These isolates were obtained from different fields in the municipality of Pinhão (25° 42 '26 "S; 51° 38' 0" W), in Paraná State. To test the pathogenicity of the isolates 20C3, 20A3, and 20A1, a conidial suspension for each isolate with 2–3 × 103 conidia per mL was sprayed at the boot stage (BBCH-scale 45) on leaves of wheat cv. Toruk and on susceptible barley cv. Ana 01 plants growing in pots. Each pot containing 5–7 plants was considered as replicate. Wheat plants were also inoculated with a P. tritici-repentis isolate with a conidial suspension at the same concentration described above to serve as reference of symptoms for tan spot. Wheat and barley plants were also inoculated with sterile water to serve as a control. Six replications were inoculated for each treatment. After inoculation, the plants were maintained in a dew chamber for 24 h and then moved to a growth room at 20±2ºC and 16 h photoperiod. Initially, the leaves of wheat plants inoculated with P. teres exhibited small, irregular, dark lesions surrounded by yellow halos, and later exhibited oval to elongate, pale brown, necrotic lesions, and yellow halos. Differently from tan spot caused by P. tritici-repentis, the wheat leaves inoculated with P. teres f. teres presented a more diffuse yellowing, and the center of necrotic lesions was pale brown. The symptoms observed in the barley leaves were the net form net blotch, which allowed us to infer that the disease was caused by P. teres f. teres isolates. All inoculated plants presented symptoms, with some variation among them. Pyrenophora teres f. teres was reisolated from the inoculated wheat and barley plants. This is the first report of P. teres f. teres causing leaf disease on wheat in Brazil. The finding contributes to a better understanding of the etiology of wheat leaf spots in humid subtropical areas of southern Brazil.
The ascomycete fungus Pyrenophora tritici-repentis is the causal agent of tan spot of wheat. The disease can occur on both common wheat (Triticum aestivum L.) and durum wheat (T. turgidum ssp. durum) and has potential to cause significant yield and quality losses. The fungal pathogen is known to produce necrotrophic effectors (NEs) that act as important virulence factors. Based on the NE production and virulence on a set of four differentials, P. tritici-repentis isolates have been classified into eight races. Race 4 produces no known NEs and is avirulent on the differentials. From a fungal collection in North Dakota, we identified several isolates that were classified as race 4. These isolates caused no or little disease on all common wheat lines including the differentials; however, they were virulent on some durum cultivars and tetraploid wheat accessions. Using two segregating tetraploid wheat populations and QTL mapping, we identified several genomic regions significantly associated with disease caused by two of these isolates, some of which have not been previously reported. This is the first report that race 4 is virulent on tetraploid wheat, likely utilizing unidentified NEs. Our findings further highlight the insufficiency of the current race classification system for P. tritici-repentis.
L’hévéa (Hevea brasiliensis) est actuellement la seule source commerciale de caoutchouc naturel. Parmi les maladies affectant l’hévéa, la CLF (« Corynespora Leaf Fall ») causée par le champignon Corynespora cassiicola, est devenue en une cinquantaine d’années un fléau pour l’ensemble des pays hévéicoles d’Asie et d’Afrique. Actuellement, la gestion du problème consiste à arracher les clones les plus sensibles et à traiter les arbres avec des fongicides en cas d’épidémie. Cependant, le nombre de clones touchés par la maladie ne cessant d’augmenter, il devient urgent de sélectionner de nouveaux clones à la fois tolérants et aptes à la production. Nos travaux ont permis de caractériser le gène codant la cassiicoline, toxine protéique glycosylée secrétée par C. cassiicola, et d’analyser sa diversité. Une étude comparative portant sur trois isolats de C. cassiicola d’agressivité contrastée a montré la présence du gène Cas1 chez les isolats de forte et moyenne agressivité, alors qu’il n’est pas détecté chez l’isolat de faible agressivité. Les niveaux d’agressivité des isolats sont corrélés aux niveaux de transcrits du gène de cassiicoline. Le rôle de la cassiicoline serait prépondérant dans les phases précoces de l’infection. L’analyse de diversité du gène de cassiicoline à partir d’une collection d’isolats provenant de différents hôtes et d’origines géographiques variées, a révélé l’existence d’au moins six isoformes protéiques (Cas1 à Cas6). La structuration génétique globale des isolats basée sur des marqueurs neutres est similaire à la structuration basée sur le gène de cassiicoline. Les isolats prélevés sur hévéa se regroupent en clades spécialisés, dont un correspondant aux isolats porteurs du gène Cas1, identifiés comme étant les plus agressifs sur hévéa. Cependant, 58 % des isolats testés semblent dépourvus de gène de cassiicoline, bien que certains génèrent des symptômes modérés sur hévéa, ce qui suggère l’existence d’autres effecteurs. Des formes endophytiques de C. cassiicola ont été isolées à partir de feuilles asymptomatiques provenant du Brésil, zone encore indemne de CLF. Les gènes de cassiicoline portés par ces souches (isoformes Cas3 et 4) ne semblent pas exprimés lors de l’interaction avec l’hévéa. Nous avons montré par ailleurs que les champignons endophytes de l’hévéa appartenant aux genres Trichoderma et Xylaria présentent une forte activité mycoparasitaire sur C. cassiicola, in vitro. Ces travaux ouvrent de nouvelles perspectives pour le contrôle de la maladie (diagnostic précoce, sélection de clones tolérants, lutte biologique).
Many species of the genus Bipolaris are important plant pathogens and often cause leaf spot, root rot, and seedling blight in an extremely wide range of hosts around the world. In recent years, maize leaf spot caused by Bipolaris species has frequently occurred with complex symptoms and is becoming increasingly serious in Sichuan Province of China. To investigate the population diversity of Bipolaris spp. and their corresponding symptoms in maize, 747 samples of maize leaf spot were collected from 132 sampling sites in 19 administrative districts of Sichuan Province from 2011 to 2018. Based on morphological characteristics, pathogenicity testing, and phylogenetic analysis of the rDNA internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, a total of 1186 Bipolaris isolates were identified as B. maydis, B. zeicola, B. cynodontis, B. oryzae, B. setariae, and B. saccharicola, among which B. maydis and B. zeicola were the dominant pathogenic species, accounting for 57.34% and 42.07% of the isolates, respectively. We found that B. zeicola isolates were mainly distributed in high altitude and cool mountainous areas, while B. maydis was more widely distributed in Sichuan Province. The typical symptoms caused by the Bipolaris species were clearly distinct in maize. The typical symptoms caused by B. maydis were elongated strip lesions, or fusiform, elliptical lesions, and those caused by B. zeicola were narrow linear lesions. Herein, B. saccharicola was first reported on maize and caused subrotund lesions. This study provides useful information for disease diagnosis and management for Bipolaris leaf spot in maize.
Drechslera leaf spot (DLS) caused by Pyrenophora (Drechslera) species is one of the most serious diseases affecting Italian ryegrass (Lolium multiflorum) in China. Between 2015 and 2018, this disease was observed in three Italian ryegrass fields in the province of Sichuan, China. Average leaf disease incidence was approximately 1 to 12% but could range up to 100%. Symptoms appeared as brown or tan spots surrounded by a yellow halo, or brown to dark brown net blotch; subsequently, spots increased in number and size, and they later covered a large area of leaf, eventually causing leaf death. In this study, 86 strains of Pyrenophora fungi were isolated from leaf lesions of Italian ryegrass. Coupled with phylogenetic analysis of the internal transcribed spacer region, partial 28S ribosomal RNA gene, and glyceraldehyde-3-phosphate dehydrogenase gene, morphological characteristics showed that Pyrenophora dictyoides and P. nobleae are associated with Italian ryegrass in southwest China. Pathogenicity tests confirmed that both species can infect Italian ryegrass, causing leaf spot, whereas the virulence of the two species differed; P. nobleae showed lower pathogenicity to Italian ryegrass. This is the first time that these two Pyrenophora species were formally reported on Italian ryegrass based on both morphological and molecular characters. Overall, this study improves knowledge of the Pyrenophora species associated with Italian ryegrass and provides a foundation for control of this disease in the future.
Neostagonosporellasichuanensis sp. nov. was found on Phyllostachysheteroclada collected from Sichuan Province in China and is introduced in a new genus Neostagonosporella gen. nov. in this paper. Evidence for the placement of the new taxon in the family Phaeosphaeriaceae is supported by morphology and phylogenetic analysis of a combined LSU, SSU, ITS and TEF 1-α DNA sequence dataset. Maximum-likelihood, maximum-parsimony and Bayesian inference phylogenetic analyses support Neostagonosporella as a distinct genus within this family. The new genus is compared with related genera of Phaeosphaeriaceae and full descriptions and illustrations are provided. Neostagonosporella is characterised by its unique suite of characters, such as multiloculate ascostromata and cylindrical to fusiform, transversely multiseptate, straight or curved ascospores, which are widest at the central cells. Conidiostromata are multiloculate, fusiform to long fusiform or rhomboid, with two types conidia; macroconidia vermiform or subcylindrical to cylindrical, transversely multiseptate, sometimes curved, almost equidistant between septa and microconidia oval, ellipsoidal or long ellipsoidal, aseptate, rounded at both ends. An updated phylogeny of the Phaeosphaeriaceae based on multigene analysis is provided.
Tan spot caused by Pyrenophora tritici-repentis is the main foliar diseases of wheat in Brazil. The effect of silicon (Si) on the components of resistance of a susceptible (Fundacep Horizonte) and a moderately resistant (Quartzo) wheat cultivar was studied in a controlled environment. Silicon was supplied as calcium silicate in the soil 30 days before sowing. At the booting stage, a conidial suspension of the fungus was sprayed onto the flag leaves of potted plants, which were incubated under moist conditions for 48 h. Afterwards, inoculated leaves were assessed for: incubation period (IP), infection efficiency (IE), area under lesion size curve (AULSC), lesion size (LS), severity (SEV) and area under severity curve (AUSC). Foliar Si concentrations were quantified at the end of the evaluations. Si supply to plants increased leaf Si concentration in 233% for Fundacep Horizonte (from 4.8 to 16.0 g kg⁻¹ of dry matter) and 211% for Quartzo (from 5.3 to 16.5 g kg⁻¹ of dry matter). In the Si + treatments, IP was longer by 24 and 17 h, IE declined by 53.5 and 65.5%, LS (at 264 h after inoculation) by 4.6 mm (from 9.5 to 4.9 mm) and 5.9 mm (from 8.2 to 2.3 mm), and SEV by 53% (from 54.4 to 18.8%) and 88% (from 47.7 to 5.5%) respectively, for the Fundacep Horizonte and Quartzo cultivars. The Si x cultivar interaction was not significant for AULSC and AUSC, and these variables were reduced by 55.8 and 80.8%, respectively, in plants supplied with Si. In conclusion, Si enhanced the resistance of wheat plants to tan spot development by affecting several resistance components, regardless of the resistance level of the cultivar. However, the greatest reduction in tan spot development by Si supply was observed when using a moderately resistant cultivar.
Genera of Phytopathogenic Fungi (GOPHY) is introduced as a new series of publications in order to provide a stable platform for the taxonomy of phytopathogenic fungi. This first paper focuses on 21 genera of phytopathogenic fungi: Bipolaris, Boeremia, Calonectria, Ceratocystis, Cladosporium, Colletotrichum, Coniella, Curvularia, Monilinia, Neofabraea, Neofusicoccum, Pilidium, Pleiochaeta, Plenodomus, Protostegia, Pseudopyricularia, Puccinia, Saccharata, Thyrostroma, Venturia and Wilsonomyces. For each genus, a morphological description and information about its pathology, distribution, hosts and disease symptoms are provided. In addition, this information is linked to primary and secondary DNA barcodes of the presently accepted species, and relevant literature. Moreover, several novelties are introduced, i.e. new genera, species and combinations, and neo-, lecto- and epitypes designated to provide a stable taxonomy. This first paper includes one new genus, 26 new species, nine new combinations, and four typifications of older names.
Bambusa pervariabilis × Dendrocalamopsis grandis blight is caused by a toxin from the fungus Arthrinium phaeospermum (corda) M. B. Ellis. We used shaking culture in a modified Fries culture medium and methanol extraction to isolate the toxin. The optimal developing solvent mixture (methanol: ethyl acetate: H2O at 7:1.5:3) was selected using thin layer chromatography and used as the eluent for toxin purification by silica gel column chromatography. Two toxic fractions were identified in the bioassay. A flaxen oil substance, AP-I, showed higher toxicity than a toxic white powder, AP-II. The more toxic AP-I was determined to be dibutyl phthalate (C16H22O4, molecular weight of 278) by mass spectrometry, nuclear magnetic resonance, and infrared spectrophotometry. Dibutyl phthalate might contribute to the pathogenesis of bamboo blight. © 2015, Northeast Forestry University and Springer-Verlag Berlin Heidelberg.
Fungi produce a diverse array of toxins that contribute both positively and negatively to agriculture. We describe phytotoxins which have defined roles in plant disease, as well as mycotoxins which generally have no direct role in disease but have significant impacts on animals that feed on infected host plants. We have included examples of host-specific phytotoxins which act upon a very narrow range of susceptible host genotypes, and non-specific phytotoxins which can affect a wide range of plants. We also describe fungal secondary metabolites that underpin certain mutualistic symbioses. Toxins are described based on their core biosynthesis, and include agriculturally important examples of non-ribosomally synthesised peptides, polyketide-based peptides, proteinaceous phytotoxins, sesquiterpenoid-containing compounds and miscellaneaous toxins of herbivores. The role of each toxin in the plant–fungus interaction, as well as the biochemistry and molecular basis of toxin biosynthesis, will be described.
Rubber tree (Hevea brasiliensis) is the only source of commercial natural rubber. The "Corynespora Leaf Fall" (CLF) disease, caused by the fungus Corynespora cassiicola, has become over the last 50 years a very serious problem in the Asian and African rubber producing countries. Currently, the main methods to face the problem are the uprooting of the most susceptible cultivars and chemical treatments in case of severe outbreaks. However, the number of cultivars affected by the disease keeps increasing, and the selection of new cultivars, both tolerant and high yielding, is urgent. In this study, the gene encoding cassiicolin, a toxic glycosylated protein secreted by C. cassiicola, was characterized and its diversity analyzed. The analysis of three C. cassiicola isolates with contrasted levels of aggressiveness revealed the presence of identical cassiicolin genes in the highly and moderately aggressive isolates but none in the isolate of mild aggressiveness. The levels of aggressiveness were correlated to the cassiicolin gene transcript levels. The cassiicolin gene was preferentially expressed in the early phase of the infection. Analysis of the cassiicolin gene diversity among isolates from various host and various geographical origins revealed the existence of at least six protein isoforms (Cas1 to Cas6). The genetic structure of the isolates based on neutral markers was closely related to the genetic structure based on the cassiicolin gene. The isolates sampled on rubber tree were grouped in several specialized clades, including one clade regrouping all the Cas1 isolates, which were the most aggressive on rubber tree. However, 58 % of the isolates seemed to be deprived of cassiicolin gene, although some of them generate moderate symptoms on rubber tree, suggesting the existence of other effectors. Endophytic C. cassiicola isolates were found in asymptomatic rubber tree leaves from Brazil, a region were CLF outbreak was never reported. No expression of the cassiicolin genes carried by these isolates could be detected in interaction with rubber tree. In addition, we found that the Trichoderma and Xylaria species, all fungal endophytes of rubber tree, were antagonists of C. cassiicola, in vitro. This work opens new perspectives for the control of CLF (through early diagnosis, selection of tolerant clones, or biocontrol).
La tache helminthosporienne, maladie du blé, communément désignée par l'appellation anglo-saxonne « tan spot » est mondialement rencontrée dans les champs de blé, et peut causer d'importantes pertes. Elle est provoquée par un ascomycète Pyrenophora tritici-repentis, dont les différents isolats semblent très variables sur le plan génétique. Cette maladie est caractérisée par des symptômes typiques, une tache nécrotique entourée d'une chlorose. Huit races de son agent causal sont actuellement reconnues, sur la base des symptômes induits (Chlorose/Nécrose) sur une gamme d'hôtes différentielle. Ces symptômes sont le résultat d'une interaction spécifique entre la plante-hôte et au moins trois toxines spécifiques à l'hôte, Ptr ToxA, Ptr ToxB et Ptr ToxC. L'analyse de cette interaction a démontré que celle-ci apparait comme l'image inversée du modèle du gène pour gène décrit par de Flore. Le présent article est une première revue bibliographique en langue française cernant les grands aspects de cette maladie, son épidémiologie et la diversité de son agent causal.
A large collaborative effort has yielded a comprehensive study of the phylogeny and a new subfamilial classification of the grass family (Poaccae/Gramineae). The study was conducted on an integrated and representative set of 62 grasses (0.6% of the species and ca. 8% of the genera) plus four outgroup taxa using six molecular sequence data sets (ndhF, rbcL, rpoC2, phyB, ITS2, and GBSSI or waxy), chloroplast restriction site data, and morphological data. A parsimony analysis using 2143 informative characters (the combined analysis) resulted in a single most parsimonious tree of 8752 steps with an RI of 0.556 and bootstrap support of < 90% for more than half of the internal nodes. Significant relationships that appear consistently in all analyses of all data sets and are strongly supported by the combined analysis include the following: Joinvilleaceae are sister to a monophyletic Poaceae; the earliest diverging linneages of the Poaceae are Anomochlooideae, Pharoideae, and Puelioideae, respectively; and all remaining grasses form a clade. Multiple monophyletic clades were recovered, including Bambusoideae s. str., Ehrhartoideae, Pooideae s.l., Aristidoideae, Danthonioideae, Chloridoideae s. str., Chloridoideae s.l., Panicoideae, Parianeae, Olyreae s. str., Oryzeae, Stipeae, Meliceae, Lygeum + Nardus, and Molinia + Phragmites. The PACCAD Clade is monophyletic, containing Aristidoideae, Danthonioideae, Arundinoideae s. str., Chloridoideae s.l., Centothecoideae, Panicoideae, Eriachne, Micraira, and Gynerium. Based on the phylogeny, a classification of II previously published subfamilies (Anomochlooideae, Pharoideae, Puelioideae, Bambusoideae, Ehrhartoideae, Pooideae, Aristidoideae, Arundinoideae, Chloridoideae, Centothecoideae, ami Panicoideae) and 1 new subfamily (Danthonioideae) is proposed. Several changes in the circumscription of traditionally recognized subfamilies are included. Previous phylogenetic work and classifications are reviewed in relation to this classification and circumscription, and major characteristics of each subfamily are discussed and described. The matrix, trees, and updated data matrix are available at 〈http://www.virtualherbarium.org/grass/gpwg/default.htm〉.
Genetic and molecular approaches were used to characterize the mating type (MAT) genes in Cochliobolus sativus. One hundred and four ascospore progeny derived from a cross of C. sativus isolates ND93-1 (MAT-1) × ND90Pr (MAT-2) were backcrossed with their parents to determine mating type, but only five progeny produced pseudothecia with asci and/or ascospores. When degenerate primers from the conserved high mobility group (HMG) protein domain encoded by the MAT-2 gene in Cochliobolus species were used in polymerase chain reaction (PCR) with genomic DNA of C. sativus as templates, an amplicon of predicted size was amplified only from MAT-2 isolates. The presence of a MAT-2 homolog in these MAT-2 isolates was confirmed by Southern hybridization with the HMG box as a probe. Additionally, the presence or absence of the HMG homolog in the progeny segregated in a 1:1 ratio, as expected for the single gene control of mating type. Using primers based on the conserved regions at the 5′ and 3′ flanks of the idiomorphs in the MAT genes of other Cochliobolus species, the full-length MAT-1 and MAT-2 idiomorphs were cloned by PCR from C. sativus isolates ND93-1 and ND90Pr, respectively. DNA sequence analysis indicated that these two idiomorphs are organized in a manner similar to their respective counterparts in other Cochliobolus species. DNA hybridization and PCR amplification analysis of 54 field isolates of C. sativus collected worldwide showed that both mating types exist in populations around the world. The low frequency of successful backcrosses of progeny to parents in the ND93-1 × ND90Pr cross, combined with the fact that many crosses between isolates of opposite mating type are unsuccessful, suggests that genetic factors other than MAT genes affect the fertility of the fungus.
Previous research indicates that infection by the tan spot fungus (Pyrenophora tritici-repentis) produces two genetically distinct symptoms in wheat (Triticum aestivum): tan necrosis and extensive chlorosis. Necrosis-inducing isolates of P. tritici-repentis release a host-selective toxin in culture that produces a reaction highly associated with the induction of tan necrosis in susceptible wheat genotypes. The objectives of this research were to determine the number of genes conditioning insensitivity to a necrosis-inducing culture filtrate in a population of wheat F-3 families, and to map the insensitivity gene(s) using restriction fragment length polymorphisms (RFLPs). The population consisted of 58 F-3 families derived from the cross of a resistant synthetic hexaploid, W-7976, with the susceptible cultivar 'Kulm.' At least 16 individuals from each F-3 family were infiltrated with culture filtrate from the P. tritici-repentis isolate 86-124 and were scored as insensitive or sensitive. Low-copy DNA clones that hybridized to group 5 wheat chromosomes were used to detect RFLPs associated with insensitivity. The families segregated in a ratio of 15:29:14 homozygous insensitive/segregating/homozygous sensitive, suggesting that a single nuclear gene was responsible for conditioning insensitivity to the pathogenic factor(s) in the culture filtrate. RFLPs were detected that flanked the locus conferring insensitivity at distances of 5.7 and 16.5 cM. Aneuploid analysis indicated that this gene resided on the long arm of chromosome 5B. We proposed the symbol tsn1 to designate this gene.
Limited black spot symptoms (Quiescent infections) develop on persimmon fruits as Alternaria alternata hyphae penetrate the pericarp of green-immature fruit. Inoculation of persimmon with A. alternata or treatment with a commercial preparation of purified cellulase induced similar black symptoms and higher peroxidase activity in green-immature fruits but not in orange-mature persimmon fruits. Both treatments induced the development of new peroxidase isoforms only in immature fruits – however, no effect was observed on polyphenoloxidase activity. A. alternata was transformed with a construct expressing green fluorescent protein (GFP). Histological analysis of hyphal development using GFP-transformed fungi indicated that symptoms are always found ahead of the leading edge of the hyphae. We suggest that peroxidase increase, induced by the directly penetrating A. alternata, might be involved in the induction of quiescence infection by the pathogen in immature fruits.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
The program MRBAYES performs Bayesian inference of
phylogeny using a variant of Markov chain Monte Carlo.
Availability: MRBAYES, including the source code, documentation,
sample data files, and an executable, is available at http://brahms.biology.rochester.edu/software.html.
Contact: johnh{at}brahms.biology.rochester.edu
The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.
The collection of papers in this issue of Mycologia documents considerable improvements in taxon sampling and phylogenetic resolution regarding the Fungal Tree of Life. The new data will stimulate new attempts to date divergences and correlate events in fungal evolution with those of other organisms. Here, we review the history of dating fungal divergences by nucleic acid variation and then use a dataset of 50 genes for 25 selected fungi, plants and animals to investigate divergence times in kingdom Fungi. In particular, we test the choice of fossil calibration points on dating divergences in fungi. At the scale of our analysis, substitution rates varied without showing significant within-lineage correlation, so we used the Langley-Fitch method in the r8s package of computer programs to estimate node ages. Different calibration points had a dramatic effect on estimated divergence dates. The estimate for the age of the Ascomycota/Basidiomycota split was 1 808 000 000 y ago when calibrated assuming that mammals and birds diverged 300 000 000 y ago, 1 489 000 000 y ago when calibrated assuming that the 400 000 000 y old fungal fossil Paleopyrenomycites devonicus represents Sordariomycetes and ~400 000 000 y ago when calibrated assuming 206 000 000 y ago for the plant eudicot/monocot divergence. An advantage of a date of ~400 000 000 y ago for the Ascomycota/Basidiomycota divergence is that the radiation of fungi associated with land plants would not greatly precede the earliest land plant fossils. Acceptance of ~400 000 000 y ago for the Ascomycota /Basidiomycota split would require that P. devonicus be considered a deeply branching Ascomycota. To improve on current estimates of divergence times, mycologists will require calibration points from within groups of fungi that share similar substitution rates. The most useful calibration is likely to depend on the discovery and description of continuous records of fossil fungi, or their spores, that show recognizable shifts in morphology.
Genetic and molecular approaches were used to characterize the mating type (MAT) genes in Cochliobolus sativus. One hundred and four ascospore progeny derived from a cross of C. sativus isolates ND93–1 (MAT-1) X ND90Pr (MAT-2) were backcrossed with their parents to determine mating type, but only five progeny produced pseudothecia with asci and/or ascospores. When degenerate primers from the conserved high mobility group (HMG) protein domain encoded by the MAT-2 gene in Cochliobolus species were used in polymerase chain reaction (PCR) with genomic DNA of C. sativus as templates, an amplicon of predicted size was amplified only from MAT-2 isolates. The presence of a MAT-2 homolog in these MAT-2 isolates was confirmed by Southern hybridization with the HMG box as a probe. Additionally, the presence or absence of the HMG homolog in the progeny segregated in a 1:1 ratio, as expected for the single gene control of mating type. Using primers based on the conserved regions at the 5’ and 3’ flanks of the idiomorphs in the MAT genes of other Cochliobolus species, the full-length MAT-1 and MAT-2 idiomorphs were cloned by PCR from C. sativus isolates ND93–1 and ND90Pr, respectively. DNA sequence analysis indicated that these two idiomorphs are organized in a manner similar to their respective counterparts in other Cochliobolus species. DNA hybridization and PCR amplification analysis of 54 field isolates of C. sativus collected worldwide showed that both mating types exist in populations around the world. The low frequency of successful backcrosses of progeny to parents in the ND93–1 X ND90Pr cross, combined with the fact that many crosses between isolates of opposite mating type are unsuccessful, suggests that genetic factors other than MAT genes affect the fertility of the fungus.
We evaluate the phylogenetic distribution of known, highly virulent plant pathogens in the genus Cochliobolus (sexually reproducing species in the Ascomycota, Pleosporaceae) and assess the relationship between Cochliobolus species and species of Curvularia and Bipolaris (asexual states of fungi in the Ascomycota, Pleosporaceae). To infer a phylogeny, we have used two sequence regions: (i) the complete ITS 1, ITS 2, and 5.8S rDNA sequences for 65 fungal isolates and (ii) a 600 bp fragment of the housekeeping gene gpd, coding for glyceraldehyde-3-phosphate dehydrogenase, for 54 isolates. We combined ITS, 5.8S and gpd sequence data from 41 species. In the Cochliobolus clade, 31 out of 32 species fit clearly into one of two groups. One species, Cochliobolus homomorphus, did not fit clearly into either group. The 13 species in Cochliobolus Group 1 grouped together with 100% bootstrap support from the combined ITS/gpd data. This group included Cochliobolus and Bipolaris species that cause serious crop losses, such as Co. sativus, Co. miyabeanus, Co. carbonum and Co. heterostrophus. However, within Group 1, the known, highly virulent pathogens did not form a monophyletic group of species. Average substitution levels between pairs of species in the Group 1 were low, about 1.7% in the ITS region, suggesting that these species had radiated rapidly and recently. The 18 species in Cochliobolus Group 2 formed a monophyletic group in 96% of parsimony bootstrap replicates of the combined ITS and gpd data. The Cochliobolus species that were transferred into the segregate genus Pseudocochliobolus were in this second group. This study included 9 Curvularia and Bipolaris species without known sexual states and they all appear to be recently derived from among sexual species of Cochliobolus. Both Curvularia and Bipolaris were polyphyletic, but only Bipolaris states were associated with Group 1 Cochliobolus species. Both Curvularia and Bipolaris states were associated with species in Cochliobolus Group 2.
The phylogeny of Pyrenophora was analyzed based on DNA sequences of two regions: the internal transcribed spacers (ITS) and a fragment (ca 600 bp) of the gpd gene coding for glyceraldehyde-3-phosphate dehydrogenase. Thirty-nine isolates of Pyrenophora/Drechslera, together with 32 isolates of five genera as outgroups were analyzed in this study. Phylogenetic analysis of the ITS and the gpd data shows that Pyrenophora is monophyletic and supports the separation of Drechslera at generic rank from other graminicolous fungi. Within the Pyrenophora clade, the sequences from multiple isolates of the same species grouped together, and asexual states clustered with their predicted sexual relatives. Overall, the relationships of most Pyrenophora/Drechslera species inferred in this study were consistent with the relationships proposed from prior morphological studies. The ITS and the gpd data sets generated in this study provided a useful database that can be used to facilitate the identification of Pyrenophora/Drechslera isolates. Eight of the 21 isolates from CBS, and three of the 17 from DOAM, labeled as Pyrenophora/Drechslera from culture collections, appeared to be misidentified. With a combination of DNA sequences and morphological data, four of these isolates were re-identified as Bipolaris species. Five isolates were re-identified within the genus Pyrenophora. One of the remaining two misidentified isolates was a Corynespora and the other remains an unidentified ascomycete. The previous transfer of Drechslera heveae and D. portulacae to Cochliobolus was supported by these molecular data.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
DNA sequences from rDNA and protein-coding regions were determined for six Embellisia and two Nimbya spp. and were compared to those from Alternaria, Ulocladium and Stemphylium spp. Sequences determined included rDNA from the nuclear internal transcribed-spacer region (ITS1/5.8S/ITS2) and the mitochondrial small-subunit (mt SSU) and a portion of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene. Phylogenetic analyses were performed on each dataset separately and then combined for total evidence analysis using methods of maximum parsimony and maximum likelihood. Results revealed that Embellisia and Nimbya clustered within a large monophyletic Alternaria-Nimbya-Embellisia-Ulocladium clade with Stemphylium as the sister taxon. Members of the infectoria species-group were the most basal group in this large polygeneric clade. Embellisia and Nimbya were sister taxa of the remaining Alternaria and Ulocladium spp. and were related more closely to Alternaria than was Stemphylium. Four Embellisia spp. formed a monophyletic clade. However, E. allii clustered with the two Nimbya spp. and E. indefessa clustered with Alternaria and Ulocladium spp., revealing that Embellisia, as currently circumscribed, is polyphyletic. Potential revisions of taxonomy for all genera are discussed.
The genus Cochliobolus (class Ascomycetes, order Pleosporales, family Pleosporaceae) contains approximately 30 species (Luttrell 1973), nearly all of which are pathogens of wild grasses or cereal crops (Sivanesan 1984). The genus is predominately heterothallic, although four homothallic species have been described (Luttrell and Rogerson 1959; Waki et al. 1979). Two genera, Bipolaris and Curvularia, have been erected for the asexual (anamorph) stages of Cochliobolus (Alcorn 1983). Before teleomorphic states of these fungi were known, anamorphic Cochliobolus spp. were described under the name Helminthosporium (Alcorn 1988). The graminicolous Helminthosporium spp. were divided into three genera as their perfect states were discovered: Bipolaris for anamorphs of Cochliobolus, Drechslera for anamorphs of Pyrenophora, Exserohilum for anamorphs of Setosphaeria.
Studies of host-specific toxins in the United States have been initiated in several laboratories, but the primary proponent of their utility to probe molecular mechanisms of fungal pathogenesis was Robert P. Scheffer. Under his influence, advances have been made in the discovery of host-specific toxins, their chemistry, their sites of action in and physiological effects on host plants, their roles in fungal pathogenesis, and the genetics of toxin production by fungi. One of his most significant contributions was to identify single genetic elements, each of which controls biosynthesis of a particular host-specific toxin. This led several investigators, after the advent of molecular genetics, to clone these elements and discover that they are large complex regions of DNA containing genes essential for toxin biosynthesis and transport. Moreover, each of these regions is unique to the genome of a particular toxin producing strain, prompting speculation that toxin biosynthesis in fungi arises as a result of horizontal gene transfer. Although it is well established that host-specific toxins contribute to the determination of fungal host range, other factors are required for general pathogenesis to plants. We have recently cloned a gene encoding a cyclic peptide synthetase, which is found in a wide array of fungal plant pathogens, and have proven by gene disruption that the hypothetical cyclic peptide is required for pathogenesis by at least two of them. Thus, it appears that some fungi exploit their capacity for production of multiple secondary metabolites to mediate their interactions with host plants.
Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces a toxin in culture that induces necrosis only on wheat cultivars susceptible to the pathogen. The toxic compound, named Ptr toxin, was purified by gel filtration chromatography and ion exchange chromatography. Unlike most other fungal phytotoxins, Ptr toxin is a low molecular weight protein of 14,700. Reduction with dithiothreitol abolished toxic activity indicating that cysteine is present and that a disulfide bond is required for activity
We evaluate the phylogenetic distribution of known, highly virulent plant pathogens in the genus Cochliobolus (sexually reproducing species in the Ascomycota, Pleosporaceae) and assess the relationship between Cochliobolus species and species of Curvularia and Bipolaris (asexual states of fungi in the Ascomycota, Pleosporaceae). To infer a phylogeny, we have used two sequence regions: (i) the complete ITS 1, ITS 2, and 5.88 rDNA sequences for 65 fungal isolates and (ii) a 600 bp fragment of the housekeeping gene gpd, coding for glyceraldehyde-3-phosphate dehydrogenase, for 54 isolates. We combined ITS, 5.8S and gpd sequence data from 41 species. Ln the Cochliobolus clade, 31 out of 32 species fit clearly into one of two groups. One species, Cochliobolus homomorphus, did not fit clearly into either group. The 13 species in Cochliobolus Group I grouped together with 100% bootstrap support from the combined ITS/gpd data. This group included Cochliobolus and Bipolaris species that cause serious crop losses, such as Co. sativus, Co. miyabeanus, Co. carbonum and Co. heterostrophus. However; within Group 1, the known, highly virulent pathogens did not form a monophyletic group of species. Average substitution levels between pairs of species in the Group 1 were low, about 1.7% in the ITS region, suggesting that these species had radiated rapidly and recently. The 18 species in Cochliobolus Group 2 formed a monophyletic group in 96% of parsimony bootstrap replicates of the combined ITS and gpd data. The Cochliobolus species that were transferred into the segregate genus Pseudocochliobolus were in this second group. This study included 9 Curvularia and Bipolaris species without known sexual states and they all appear to be recently derived from among sexual species of Cochliobolus. Both Curvularia and Bipolaris were polyphyletic, but only Bipolaris states were associated with Group 1 Cochliobolus species. Both Curvularia and Bipolaris states were associated with species in Cochliobolus Group 2.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
The phylogeny of Pyrenophora was analyzed based on DNA sequences of two regions: the internal transcribed spacers (ITS) and a fragment (ca 600 bp) of the gpd gene coding for glyceraldehyde-3-phosphate dehydrogenase. Thirty-nine isolates of Pyrenophora/Drechslera, together with 32 isolates of five genera as outgroups were analyzed in this study. Phylogenetic analysis of the ITS and the gpd data shows that Pyrenophora is monophyletic and supports the separation of Drechslera at generic rank from other graminicolous fungi. Within the Pyrenophora clade, the sequences from multiple isolates of the same species grouped together, and asexual states clustered with their predicted sexual relatives. Overall, the relationships of most Pyrenophora/Drechslera species inferred in this study were consistent with the relationships proposed from prior morphological studies. The ITS and the gpd data sets generated in this study provided a useful database that can be used to facilitate the identification of Pyrenophora/Drechslera isolates. Eight of the 21 isolates from CBS, and three of the 17 from DOAM, labeled as Pyrenophora/Drechslera from culture collections, appeared to be misidentified. With a combination of DNA sequences and morphological data, four of these isolates were re-identified as Bipolaris species. Five isolates were re-identified within the genus Pyrenophora. One of the remaining two misidentified isolates was a Corynespora and the other remains an unidentified ascomycete. The previous transfer of Drechslera heveae and D. portulacae to Cochliobolus was supported by these molecular data.
The resistance of strains of northern, southern, and northern/southern hybrids of smooth bromegrass (Bromus inermis) to Pyrenophora bromi, the cause of a major leaf spot disease, was determined in field tests in northern Saskatchewan from 1967 to 1971. Infection derived from natural epidemics was supplemented with that from highly susceptible infector plants spaced in the test plots to improve uniformity of inoculum supply. No bromegrass strains were completely resistant to the pathogen. Four of the eight northern/southern cultivars and three of the five southern cultivars were more resistant to P. bromi than any of the nine northern types. The best southern cultivars, Baylor and Sac, were not significantly more resistant than the cult Magna and less resistant than the S-7382 and S-7306 strains, all of northern/southern-type. Basic clones of northern/southern-type strains, adapted to Western Canada, were superior in resistance to northern clones. Parental clones were more resistant than their progeny and this resistance declined from the Syn. 1 to the Syn. 2 in two northern/southern strains. Selection for resistance to the other major leaf spot pathogen, Selenophoma bromigena, resulted in a limited improvement also in resistance to P. bromi.
Smooth bromegrass (Bromus inermis Leysser) germplasm PL-BDR1 and two seed increase generations derived from it are highly resistant to brown leaf spot caused by Pyrenophora bromi (Died.) Drechs. [Drechslera bromi (Died.) Shoemaker, conidial state = Helminthosporium bromi Died.)]. Lesions are about one-half the width and one-tenth the area of spots on susceptible cultivars. Physiological or anatomical mechanisms of resistance have not been identified, but they could have a bearing on stability of resistance and on forage quality components. This study was conducted to determine whether certain resistance mechanisms identified in other leaf spot diseases of grasses operate in PL-BDR1 germplasm. The three resistant generations were compared with susceptible cultivars Belmont, Radisson, and Saratoga for fungal penetration, halo formation, and leaf vein architecture. Susceptible leaves supported higher numbers of penetration attempts and successful penetrations. No effect of plant line was found for appositional wall formation (= papilla formation), initial colonization rate, distance between parallel veins, proportion of large vascular bundles, or girdering of vascular bundles. Resistant lines had wider vascular bundles than susceptible cultivars indicating a possible role for strength of the vascular framework in restricting lateral spread. Extensive cell necrosis and chlorotic halo formation in susceptible cultivars indicated possible involvement of a fungal phytotoxin. Resistant lines had few halos, and reduced sensitivity to toxins may be involved in resistance of PL-BDR1 to brown leaf spot.
Smooth bromegrass ( Bromus inermis Leyss.) cultivars are susceptible to brown leaf spot ( Pyrenophora bromi [Died.] Drechs.); however, some plants have smaller lesions than others. We studied inheritance of lesion size because there was no previous information on inheritance of resistance to brown leaf spot. Ten parent clones differing in lesion size were reciprocally single crossed with parent clone 11. The 10 parental clones were also polycrossed. Greenhouse‐grown parents and progeny were inoculated with conidia of P. bromi and rated for lesion size in November 1979. Regrowth was inoculated and rated in January 1980. Reciprocals were not statistically different. Four parental clones were more resistant than parent 11 in at least one of the two trials, but the F 1 progeny of two of these clones were significantly more susceptible than the midparent (mean of clone 11 and the single cross parent). Five other parental clones were more susceptible than clone 11, and F 1 progeny of two of them were more susceptible than the midparent. Polycross progeny means did not differ from polycross midparent means or were slightly lower in 2 of 20 observations. The results indicated that lesion size is regulated by multiple genes, and that susceptibility may be dominant or epistatic to resistance. There was a significant entry ✕ date interaction believed to be attributable to environment before and after inoculation. Developing populations with high levels of resistance to brown leaf spot will be difficult.
Tan spot, caused by Pyrenophora tritici-repentis, is an important foliar disease of wheat worldwide. The fungus produces two distinct symptoms, necrosis (nec) and chlorosis (chl), on susceptible wheat genotypes. Fungal isolates have been grouped into five races based on their ability to induce necrosis and/or chlorosis on differentials Glenlea, Katepwa, 6B365, and Salamouni (1). Moreover, the isolates were designated on their ability to induce necrosis and chlorosis as follows: nec+chl+ (necrosis and chlorosis), nec+chl- (necrosis only), nec-chl+ (chlorosis only), and nec-chl- (neither symptom). Races 3 and 5 induce extensive chlorosis (nec-chl+) on 6B365 and Katepwa, respectively. Race 5 was reported on durum from North Africa. Races 1 to 4 were described from North America (1,2). During 1998, a survey of durum fields was conducted in the primary durum-growing area of North Dakota to assess the virulence pattern of P. tritici-repentis. Fifty-two single-spore isolates were obtained from diseased leaves. The isolates were evaluated for their virulence by inoculating them individually onto 15 seedlings of each wheat differential in the greenhouse. Forty-nine of 52 isolates were grouped as race 1 (nec+chl+) and three isolates, obtained from the Langdon Experiment Research Station, were grouped as race 5 (nec-chl+). Race 5 isolates were evaluated three times and consistently induced extensive chlorosis on Katepwa. This is the first report of the occurrence of race 5 outside of North Africa. This race may threaten wheat in the United States, so cultivars and germplasm should be evaluated for resistance. More isolates are under investigation to obtain a comprehensive virulence pattern of the pathogen population in the United States.
References: L. Lamari and C. C. Bernier. Can. J. Plant Pathol. 11:49, 1989; (2) L. Lamari et al. Can. J. Plant Pathol. 17:312, 1995
A study was undertaken to determine the distribution of Pyrenophora tritici-repentis and Leptosphaeria nodorum, two foliar pathogens of wheat, on smooth bromegrass (Bromus inermis), an alternative host. Smooth bromegrass was selected for evaluation as a host for foliar pathogens of wheat because it is widespread along roadways and windbreaks near fields of cereal crops. From 1981 through 1984, leaf samples were collected in the northern Great Plains, including 125 in North Dakota, 32 in South Dakota, 24 in Minnesota, and 27 in Montana. Of the 208 smooth bromegrass samples collected, 70% were infected with L. nodorum, 59% with Pyrenophora spp., 52% with Pseudoseptoria bromigena, and 46% with Cochliobolus sativus. Of the 71 isolates of Pyrenophora spp. obtained, 52 were P. tritici-repentis and 19 were P. bromi. The number of P. bromi cultures isolated from the 19811983 collections was low because the primary objective was to obtain isolates of P. tritici-repentis. P. tritici-repentis and L. nodorum were widely distributed throughout the northern Great Plains on smooth bromegrass. Thus smooth bromegrass is a good alternative host for L. nodorum and P. tritici-repentis and could provide inoculum for cereal crops planted in the northern Great Plains. Mycelium growth rate on sucrose-proline agar and spore production on lima bean agar were useful in separating P. tritici-repentis from P. bromi.
Tan spot of wheat, caused by the ascomycete Pyrenophora tritici-repentis, is an economically important disease in all the major wheat growing areas worldwide. Even though the pathogen was known to occur on grasses and episodically on wheat for more than eight decades, large-scale epidemics of tan spot were first recorded in the early 1970s. The increased incidence was associated with stubble retention, a practice implemented in the context of soil conservation. The present review highlights some of the recent developments that have occurred in studies of the wheat - P. tritici-repentis interaction and discusses the implications for our understanding of host-parasite relations in general. Races of P. tritici-repentis produce at least three host-specific toxins, effective on particular host lines or cultivars. Single dominant and independently inherited genes control host reaction to these toxins, with one gene for each toxin. Eight races of the pathogen have now been identified from collections made in several parts of the world, accounting for all virulence patterns expected from three toxins matching three "susceptibility" genes in the host. Genetic analyses of host and pathogen suggested that a one-to-one relationship existed in the wheat - P. tritici-repentis interaction. The model described for tan spot of wheat appears to be a mirror image of the classical gene-for-gene in that it is based on compatibility. Thus, it is proposed that the gene-for-gene model can be extended, as previously predicted by others, to pathosystems involving multiple host-specific toxins. The relationship between toxin production and the evolution of new races is also discussed.
Pyrenophora tritici-repentis, the causal agent of tan spot of wheat, differentially induces tan necrosis and/or chlorosis in wheat. A chlorosis-inducing, host-specific toxin, termed Ptr ToxB (formerly Ptr chlorosis toxin), was purified from the culture filtrates of a race 5 isolate of P. tritici-repentis. Partial purification was performed by 25 to 80% ammonium sulfate precipitation and passage through a carboxymethyl-Sephadex C-25 cation exchange column. Final purification was performed by fast performance liquid chromatography, with a Mono S HR 5/5 cation exchanger, followed by size fractionation on a Superose 12 HR 10/30 column. The toxin was shown to be proteinaceous in nature, and purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of Ptr ToxB was determined to be 6.61 kDa. The amino acid composition and partial N terminus amino acid sequence of the toxin were also obtained. Ptr ToxB was found to be heat stable, maintaining full toxic activity after 1 h at 55°C. Infiltration of toxin concentrations as low as 14 nM produced chlorosis on susceptible cultivars.
Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces Ptr ToxB, a host-specific protein toxin encoded by the ToxB gene. Three of an estimated 8–10 copies of ToxB from an Algerian race 5 isolate (Ptr ToxB+) of the pathogen were cloned and characterized, as was a ToxB homolog identified in a race 3 isolate from Canada that lacks Ptr ToxB toxin activity. All three loci from the race 5 isolate possessed identical 261-bp open reading frames (ORFs) and were associated with retrotransposon-like sequences; the ToxB ORF from the race 3 isolate was nearly identical, but the upstream flanking region was unique to this locus. The presence of ToxB homologs was also confirmed in Canadian isolates of races 4 and 5 showing reduced or no toxin activity. ToxB was found to be expressed in mycelia and conidia of the Algerian race 5 isolate, but only in conidia of the Canadian races 3, 4 and 5 isolates, indicating differences in transcriptional regulation. Comparison of the sequence information obtained in this study with previous reports suggested that the ToxB gene is highly conserved across a geographically-diverse set of isolates.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Discussions among researchers working on the Pyrenophora tritici-repentis/wheat interaction have led to a proposal for the standardization of the nomenclature for the host-selective toxins (HSTs) produced by this fungus. Advances in the isolation and purification of the toxin(s) produced by P. tritici-repentis and the likelihood that this pathogen produces multiple toxins necessitate a systematic approach to naming these molecules and the genes that direct their production. Presently, there is one HST that has three names and two apparently distinct HSTs that are informally referred to by the same name. To improve communication among the researchers working in this area and with those interested in the literature, we propose a system of nomenclature for the HSTs produced by this pathogen and genes responsible for their production.
A total of 695 wheat accessions were evaluated for reaction to P. tritici-repentis in the growth room and categorized, using a rating system based exclusively on lesion type. A range of reactions was observed in the di-, tetra-, hexa- and octoploid wheats, and high levels of resistance were identified in all ploidy groups. Lesion type did not change with increasing period of post-inoculation leaf wetness. The wild wheat species had generally higher proportions of resistant genotypes than the cultivated tetra- and hexaploid species. Resistance was characterized by small dark brown to black spots without, or with slight amounts of, tan necrosis or chlorosis; and susceptibility was characterized by either tan necrosis or chlorosis, with or without small dark brown to black spots. About 5% of the entries in all of the ploidy groups, except the octoploid, developed small brown spots and extensive chlorosis that expanded to cover the entire leaf. Reactions obtained in tests at the 2-leaf stage were similar to those obtained at the 4 - to 6-leaf stage in the growth room and on mature plants in the field. Cultivars with the lowest scores for lesion type had the lowest ratings for disease severity in artificially inoculated field plots and in plots sown into infested wheat stubble.
Tan necrosis and extensive chlorosis are two distinct symptoms induced by Pyrenophora tritici-repentis in susceptible hexaploid wheats (Triticum aestivum). To date, five races of the pathogen have been identified based on virulence patterns on a differential set of wheat genotypes. The inheritance of wheat reaction to three races of P. tritici-repentis, races 2, 3 and 5, and to the toxins Ptr ToxA and Ptr ToxB produced by races 2 and 5, respectively, was determined. Race 2 and Ptr ToxA induce necrosis on susceptible host plants while race 3, race 5, and Ptr ToxB induce chlorosis. Crosses were made among seven hexaploid wheat genotypes and the F1, F2, BC1F1, and BC1F2 progeny were evaluated for reaction to the appropriate races and/or pathogen-produced toxins. No segregation was observed in all crosses between resistant lines. Three independently inherited loci were identified, each locus controlling the reaction to a single race. Susceptibility (necrosis or chlorosis) was dominant for all three loci identified in this study. Sensitivity to Ptr ToxB produced by race 5 and susceptibility to race 5 were found to be controlled by the same locus.
We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a ‘universal’ primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the ‘universal’ ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.
Host-selective toxins are known determinants of compatibility in plant–fungus interactions and provide a powerful model for understanding the specificity of these associations. The identification of genes required for toxin biosynthesis has shown that the genes are unique to the toxin producing species and are clustered in complex loci. These loci may have been acquired by horizontal gene transfer. Many, if not all, host-selective toxins act by disrupting biochemical processes and in several cases the resulting cell death has the characteristics of programmed cell death. This ability to make dead tissue from living has enabled these facultative saprophytic fungi to become plant pathogens.
The Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide, host-specific toxin, AM-toxin. We recently cloned a cyclic peptide synthetase gene, AMT, whose product catalyzes the production of AM-toxin and showed that it resides on chromosomes of 1.8 Mb or less, depending on the A. alternata apple pathotype strain. Reverse transcriptase (RT)-PCR, using primers specific to AMT, on laboratory sub-cultured strains previously shown to produce AM-toxin, identified one isolate that did not express the gene. A leaf necrosis bioassay confirmed an AM-toxin-minus phenotype. However, an original isolate of this strain which had not undergone sub-culture gave a positive result by both RT-PCR and bioassay. Contour-clamped homogeneous electric field electrophoresis and Southern hybridization demonstrated the loss of a 1.1-Mb chromosome in the non-toxin-producing isolate. Since this chromosome can be entirely lost without affecting growth, but is necessary for pathogenicity, we propose it is a conditionally dispensable chromosome.
Isolates of Pyrenophora tritici-repentis, the causal agent of tan spot, cause necrosis only, chlorosis only or necrosis and chlorosis as symptoms of infection on appropriate wheat differentials. A toxin has been purified from a culture filtrate of a necrosis-only isolate. The toxin produces necrosis if it is infiltrated into the leaves of cultivars susceptible to the isolate but is without effect on cultivars resistant to the isolate. The toxin was purified by gel filtration and ion exchange chromatography and shown to be a protein. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to show the toxin purity as well as estimate a protein molecular weight of 13900. The amino acid composition and molar extinction were determined. Proteolytic digestion or treatment of the purified toxin under denaturing conditions destroyed toxin activity. Infiltration of 10−13-10−14 moles of purified toxin into susceptible cultivars will produce necrosis.
