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Outbreak of Leaf Spot and Fruit Rot in Florida Strawberry Caused by Neopestalotiopsis spp
Abstract
Pestalotiopsis-like species have been reported affecting strawberry worldwide. Recently, severe and unprecedented outbreaks have been reported in Florida commercial fields where leaf, fruit, petiole, crown, and root symptoms were observed, and yield was severely affected. The taxonomic status of the fungus is confusing since it has gone through multiple reclassifications over the years. Morphological characteristics, phylogenetic analyses, and pathogenicity tests were evaluated for strawberry isolates recovered from diseased plants in Florida. Phylogenetic analyses derived from the combined ITS, β-tub, and tef1 regions demonstrated that although there was low genetic diversity among the strawberry isolates, there was a clear separation of the isolates in two groups. The first group included isolates recovered over a period of several years, which was identified as Neopestalotiopsis rosae. Most isolates recovered during the recent outbreaks were genetically different and may belong to a new species. On PDA, both groups produced white, circular, and cottony colonies. From the bottom, colonies were white to pale yellow for Neopestalotiopsis sp. and pale luteous to orange for N. rosae. Spores for both groups were five-celled with three median versicolored cells. Mycelial growth and spore production were higher for the new Neopestalotiopsis sp. isolates. Isolates from both groups were pathogenic to strawberry roots and crowns. However, the new Neopestalotiopsis sp. proved more aggressive in fruit and leaf inoculation tests, confirming observations from the recent outbreaks in commercial strawberry fields in Florida.
... Strawberry (Fragaria × ananassa Duchesne ex Rozier) production is persistently threatened by a broad spectrum of fungal pathogens including Colletotrichum spp., Phytophthora cactorum, and emerging Neopestalotiopsis species that are causal agents of fruit, root, and crown rots (Baggio et al., 2021;Jiménez et al., 2023;N.-Y. Wang et al., 2019). ...
... The first report of these pathogens in strawberry arose from a fruit rot outbreak in Central Florida in 1972 (Howard, 1973), and have since been reported primarily causing root and crown rots in Egypt, Spain, Mexico, and the United States, among other regions (Chamorro et al., 2016;Essa et al., 2018;Rebollar-Alviter et al., 2020). Despite their spread, these pathogens had not yet posed a serious threat to strawberry production and were rather considered secondary pathogens that infect the roots and crowns of weakened hosts (Baggio et al., 2021). However, in 2017, Neopestalotiopsis-like isolates were recovered from lesions on strawberry leaves and fruit in a severely impacted production field in Central Florida (Baggio et al., 2021). ...
... Despite their spread, these pathogens had not yet posed a serious threat to strawberry production and were rather considered secondary pathogens that infect the roots and crowns of weakened hosts (Baggio et al., 2021). However, in 2017, Neopestalotiopsis-like isolates were recovered from lesions on strawberry leaves and fruit in a severely impacted production field in Central Florida (Baggio et al., 2021). These isolates were grouped within a phylogeny of Neopestalotiopsis based on the internal transcribed spacer (ITS), β-tubulin (β-tub), and translation elongation factor-1 alpha (tef1) sequences but were distinct from strawberry isolates recovered prior to 2017 (Baggio et al., 2021). ...
Aggressive strains of Neopestalotiopsis sp. have recently emerged as devastating pathogens of strawberry (Fragaria × ananassa Duchesne ex Rozier), infecting nearly all plant parts and causing severe outbreaks of leaf spot and fruit rot in Florida and globally. The development of host resistance is imperative due to the absence of fungicides that effectively inhibit Neopestalotiopsis sp. growth on an infected strawberry crop. Here, we analyzed 1578 individuals from the University of Florida's (UF) strawberry breeding program to identify and dissect genetic variation for resistance to Neopestalotiopsis sp. and to explore the feasibility of genomic selection. We found that less than 12% of elite UF germplasm exhibited resistance, with narrow‐sense heritability estimates ranging from 0.28 to 0.69. Through genome‐wide association studies (GWAS), we identified two loci accounting for 7%–16% of phenotypic variance across four trials and 3 years. Several candidate genes encoding pattern recognition receptors, intra‐cellular nucleotide‐binding leucine‐rich repeats, and downstream components of plant defense pathways co‐localized with the Neopestalotiopsis sp. resistance loci. Interestingly, favorable alleles at the largest‐effect locus were rare in elite UF material and had previously been unintentionally introduced from an exotic cultivar. The array‐based markers and candidate genes described herein provide the foundation for targeting this locus through marker‐assisted selection. The predictive abilities of genomic selection models, with and without explicitly modeling peak GWAS markers as fixed effects, ranged between 0.25 and 0.59, suggesting that genomic selection holds promise for enhancing resistance to Neopestalotiopsis sp. in strawberry.
... (Baggio et al. 2019a;Irzykowska et al. 2005;Koike and Gordon 2015). However, Neopestalotiopsis species have emerged as potent fungal pathogens causing root and crown rot, leaf spot and blight, and fruit rot (Baggio et al. 2021;Chamorro et al. 2016;Rebollar-Alviter et al. 2020;Shi et al. 2022). Although these symptoms are so far mainly associated with Neopestalotiopsis rosae (Essa et al. 2018;Rebollar-Alviter et al. 2020;Sun et al. 2021), other Neopestalotiopsis spp., such as N. clavispora (Shi et al. 2022;Sigillo et al. 2020), N. mesopotamica, N. iranensis (Ayoubi and Soleimani 2016;Hidrobo-Chavez et al. 2022;Intriago-Reyna et al. 2021), and N. fragariae (Prematunga et al. 2022), have also been reported in strawberry commercial production fields. ...
... Based on field observations, it is known that this fungus spreads over long distances via the distribution of infected plants from nurseries. Once in commercial fields, the disease (leaf spot and leaf blight phases) spreads mainly by rain splashing onto and from infected leaves and other vegetative parts (Baggio et al. 2021;Rebollar-Alviter et al. 2020). Although the leaf spot and leaf blight phases of the disease could be crucial for open-field production systems, such as in Florida, U.S.A., under protected conditions (e.g., high tunnel systems), these symptoms are likely not a concern because the splash-dispersed nature of the disease is significantly reduced by plastic covering of the crop. ...
... Since then, the disease has become the dominant concern of the strawberry industry because of its high destructive capacity and the possibility of being associated with other crown rot pathogens, such as F. oxysporum f. sp. fragariae, M. phaseolina, and Ilyonectria destructans (Baggio et al. 2021;Lieten et al. 2021). ...
Strawberry root and crown rot caused by the fungus Neopestalotiopsis rosae is an emerging disease that has caused yield losses reaching 70% in Mexico and other regions worldwide. This research evaluated the effects of biological and chemical fungicides applied as preventive and curative applications for controlling root and crown rot caused by N. rosae in strawberries under greenhouse conditions. Treatments included these chemical fungicides: prochloraz, prochloraz+thiram, cyprodinil+fludioxonil, difenoconazole+azoxystrobin, iprodione, captan, thiram, pydiflumetofen+fludioxonil, fluxapyroxad+pyraclostrobin, and hymexazol; each applied at commercial doses. Also tested were biological treatments based on Trichoderma koningiopsis, Trichoderma asperellum, Streptomyces sp., and Bacillus amyloliquefaciens strain D747 (B. velezensis). Disease incidence, severity, plant mortality, root length, and dry weight were determined. Results showed that overall, preventive applications of the fungicides pydiflumetofen+fludioxonil, cyprodinil+fludioxonil, and prochloraz resulted in the smallest area under the disease progress curve, and lowest final disease incidence, severity, and plant mortality. An intermediate group of effective treatments entailed hymexazol, iprodione, T. asperellum, and T. koningiopsis (50–75% efficacy). Treatments with greater efficacy (99 to 100%), prochloraz pydiflumetofen+fludioxonil, cyprodinil+fludioxonil, and prochloraz, also had maximal total plant biomass vis-à-vis the untreated control. In contrast, each treatment’s efficacy was significantly reduced when applied curatively (0 to 37% treatment efficacy). These results suggest that certain treatments are useful for controlling strawberry root and crown rot caused by N. rosae, when applied preventively (as root dipping). These results will contribute to design more effective management programs of root rot and crown rot caused by N. rosae on strawberry.
... However, strawberry plants are susceptible to many pathogens, particularly fungi. Pestalotiopsis longisetula was first reported in 1972, causing fruit rot (Baggio et al. 2021). In 2017, a Pestalotiopsis-like fungus was first observed in Florida commercial strawberry fields, causing leaf, fruit, crown, and root symptoms. ...
... genetically close to N. rosae but significantly more aggressive. It caused an unprecedented and severe outbreak in the 2019 to 2020 season and has become an emerging issue in Florida (Baggio et al. 2021). Although the genome of N. rosae has recently become available (Hsu et al. 2022), a chromosome-scale reference genome has not been reported. ...
... Two genome assemblies representing two isolates with different pathogenicity to strawberries (Fragaria × ananassa) were assembled to determine possible genomic structure variations associated with pathogen virulence. The Neopestalotiopsis isolates used in this study, 19-02 and 13-481, were collected from the diagnostic clinic at the Gulf Coast Research and Education Center at the University of Florida (Baggio et al. 2021). Isolate 13-481 collected from roots of 'Strawberry Festival' (Fragaria × ananassa) (Chandler et al. 2000) exhibited moderate virulence and was identified as N. rosae based on both molecular post-PCR high-resolution melting and phylogenetic analysis using sequences for internal transcribed spacer (ITS), beta tubulin (β-tub), and translation elongation factor (tef1) ). ...
Several Neopestalotiopsis spp. have been reported to affect strawberry worldwide. Here, we sequenced two isolates of Neopestalotiopsis 19-02 (highly virulent) and 13-481 (moderately virulent) recovered from commercial strawberry fields in Florida and generated chromosome-scale assemblies using the Oxford Nanopore Technologies and Illumina platforms. Each genome assembly for 19-02 and 13-481 contained putative telomere sequences at the 5′ and 3′ ends. By analyzing the collinearity of the two newly assembled genomes and verifying the conserved telomere sequence at the end of each chromosome, we confirmed that the genome of Neopestalotiopsis spp. consists of seven basic chromosomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed 98.5% of conserved core genes for both genome assemblies, 19-02 and 13-481. Phylogenetic analysis with internal transcribed spacer, beta-tubulin, and translation elongation factor regions obtained from the assemblies 19-02 and 13-481 demonstrated that the highly virulent isolate 19-02 is phylogenetically distinct from moderate virulent N. rosae 13-481. Comparative genome analysis of the high-quality reference genome assemblies provided in this study will facilitate the identification of genomic regions responsible for the different virulence of the Neopestalotiopsis pathogen to strawberry.
[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.
... In Indonesia, Neopestalotiopsis has not been reported as plant pathogen yet, however in many countries this pathogen was reported as a causal agent for many important diseases. An outbreak of strawberry leaf spot and fruit rot caused by Neopestalotiopsis spp. was reported in Florida (Baggio et al., 2021;Baggio and Peres, 2020). Neopestalotiopsis siciliana sp. ...
... Neopestalotiopsis pathogens are often considered as weak pathogen that infects plants with weak conditions. However in Florida, N. rosae on strawberry plants showed much higher infection severity which the symptoms appeared in several types of tissues (Baggio et al., 2021;Baggio and Peres, 2020). In the case of strawberry plants, Neopestalotiopsis leaf spot disease symptoms are similar to leaf scorch disease and ordinary leaf spot. ...
Leatherleaf fern ( Rumohra adiantiformis ) is an important ornamental plant in Indonesia and global. Green fern leaves with bold dark green color with long shelf-life, attract florists as decoration. Indonesia is one important leatherleaf fern exporters, however currently an outbreak of leaf blight decreased production significantly. Initial symptom was reddish brown spots from edge of leaf, which was gradually followed by dark-brown necrotic lesions causing leaf blight and dried. This is a study to do Koch-Postulate approach and molecular identification, to identify the pathogen of the “new emerging disease” reported. Based on multigene analysis using primers from ITS, β-tub and tef1-α gene markers, the pathogen was identified as Neopestalotiopsis sp. All sequences have been deposited in GenBank with accession number of OR905551 (ITS), OR899817 (ß-tubulin) and OR899816 (TEF). This Neopestalotiopsis leaf blight causes an emerging concern in leatherleaf fern in Indonesia and global biosecurity because it infected an export commodity.
... Following two days of incubation at 24 • C, single hyphal tips were transferred to potato dextrose agar and incu- four cultures shared 100% nucleotide identity. The genomic sequence data shared 100% nucleotide identity with the aggressive Neopestalotiopsis species that was first reported in North America (Baggio et al., 2021). Sequence identities include 375/375 bp for the ITS, 381/381 bp β-tubulin, and 588/588 bp for the translation elongation factor 1-alpha. ...
... The DNA sequence from all three loci had 100% sequence identity to the original ITS, β-tubulin, and translation elongation factor 1-alpha loci. This is the first time that the aggressive species of Neopestalotiopsis, previously reported in the United States (Baggio et al., 2021;Rotondo et al., 2022;Guan et al., 2023) has been reported in Canada. ...
... Currently over 70 species of Neopestalotiopsis are listed in Index Fungorum (2022). Diseases caused by Neopestalotiopsis species in Taiwan as well as globally are an emerging issue (Ayoubi and Pari 2016;Ayoubi and Soleimani 2016;Baggio et al. 2021;Chamorro et al. 2016;Hsu et al. 2020;Jayawardena et al. 2015;Liu et al. 2017;Machín et al. 2019;Obregón et al. 2018;Sun et al. 2021;Tsai et al. 2021). Recent studies done by our group and other researchers found that Neopestalotiopsis species are associated with numerous diseases in various hosts such as tea ) and strawberry in Taiwan. ...
... The infection became predominant and more severe during 2019 to 2020, causing up to 30% plant loss after transplanting ). Subsequently, the same species was recently reported in Florida by Baggio et al. (2021), devastating strawberry fields in the United States and thus becoming a serious issue for the strawberry industry. The development of whole-genome sequencing technology and successive application of the Nanopore and Illumina approaches can produce genome assemblies for various important pathogenic fungi (Grigoriev et al. 2013). ...
Neopestalotiopsis rosae is one of the most harmful pathogenic fungi for strawberry, causing leaf blight and crown rot, and has become the main strawberry disease worldwide. In this study, we present the first high-quality genome of N. rosae strain ML1664, combining Nanopore long-read and Illumina short-read sequencing technologies. A total of 18 contigs were assembled with a genome size of 53.78 Mbp, predicting 15,966 putative protein-coding genes and 1,822 candidate secreted proteins in the N. rosae ML1664 genome. The high-quality genome of N. rosae will enhance our understanding regarding the biology and genetics of this pathogen. Furthermore, our resources will provide valuable information for studying the infection process, pathogen virulence, and management strategies of N. rosae.
... Our strain AC50 has identical ITS and TEF1 and highly similar TUB2 (99.8%; 1 nt difference) sequences to the type strain of N. rosae (CBS 101057). Neopestalotiopsis rosae has been recorded as a pathogen of various fruit crops, e.g., blueberry (Vaccinium corymbosum; [53,63]), pomegranate (Punica granatum; [64]), and in particular strawberry (Fragaria × ananassa), on which it was reported to cause severe disease outbreaks around the world in recent years (e.g., Australia [65], China [66], Mexico [52], Taiwan [67], and the USA [65]). In the protologue of N. rosae, it was characterized by three to five tubular apical appendages not arising from the apical crest but at different regions in the upper half of the apical cell. ...
... Our strain AC50 has identical ITS and TEF1 and highly similar TUB2 (99.8%; 1 nt difference) sequences to the type strain of N. rosae (CBS 101057). Neopestalotiopsis rosae has been recorded as a pathogen of various fruit crops, e.g., blueberry (Vaccinium corymbosum; [53,63]), pomegranate (Punica granatum; [64]), and in particular strawberry (Fragaria × ananassa), on which it was reported to cause severe disease outbreaks around the world in recent years (e.g., Australia [65], China [66], Mexico [52], Taiwan [67], and the USA [65]). In the protologue of N. rosae, it was characterized by three to five tubular apical appendages not arising from the apical crest but at different regions in the upper half of the apical cell. ...
Avocado (Persea americana) represents an important emerging tropical crop in Italy, espe- cially in the southern regions. In this study, young plants of avocado showing symptoms of stem and wood lesion, and dieback, were investigated. Isolations from symptomatic tissues consistently yielded colonies of Neopestalotiopsis-like species. The characterization of representative isolates was based on the observation of morphological characters, the effect of temperature on mycelial growth rate, and on the sequencing of three different gene regions, specifically ITS, TEF1, and TUB2. Phyloge- netic analyses were conducted based on maximum parsimony and maximum likelihood approaches. The results showed the presence of two species, viz. Neopestalotiopsis rosae and N. siciliana, the latter of which is here described as a new species. Pathogenicity tests were conducted using the mycelial plug technique on young potted avocado trees for both Neopestalotiopsis species. The results showed that both species were pathogenic to avocado. This study represents the first report of these two species affecting avocado and results in the description of a new species within the genus Neopestalotiopsis. Based on phylogeny, Pestalotiopsis coffeae-arabicae is combined in Neopestalotiopsis.
... causing strawberry fruit rot. (Baggio et al. 2021a;Kaur et al. 2022). ...
Florida’s strawberry industry is currently valued at $511 million annually but faces challenges from pathogens and arthropod pests especially Tetranychus urticae Koch (twospotted spider mite) and Scirtothrips dorsalis Hood (chilli thrips). Predatory mites, particularly Neoseiulus cucumeris Oudemans, Neoseiulus californicus McGregor, and Amblyseius swirskii Athias-Henriot, play a crucial role in pest management. However, there are concerns regarding how these biological control agents are affected by fungicides used in current pathogen management strategies. This study assessed the residual effects of commonly used fungicides in strawberries on the survival, feeding, and oviposition of these predatory mites. Commercially sourced predatory mites were reared on S. dorsalis larvae, and gravid female predators placed on fungicide treated strawberry leaf discs in a Munger cell for 120 h. Fungicides tested included two formulations of Captan, hydrogen peroxide + peroxyacetic acid, cyprodinil + fludioxonil, tetramethylthiuram disulfide, cyflufenamid and a control. All fungicides tested had an impact on the survival, feeding, and oviposition of the predators. Among the fungicide treatments, the lowest predator survival was observed in the cyprodinil + fludioxonil treatment, while the highest was observed in the hydrogen peroxide + peroxyacetic acid and tetramethylthiuram disulfide treatments. In all treatments, feeding and oviposition greatly varied among predators; specifically, N. cucumeris and A. swirskii had the lowest prey consumption, while N. californicus had the highest. These findings highlight the potential incompatibility between fungicides and predatory mites and demonstrate the need for the development of a fungicide rotation program tailored to the different susceptibilities of predators to fungicides.
... Anthracnose (caused by Colletotrichum fragariae) can be a severe problem in some years when conditions are conducive, and the disease was established during plant production. The new virulent strain of Neopestalotiopsis that recently emerged in Florida (Baggio et al., 2021) has not been found in California. ...
Fruit production, plant production, genetics, technological advancements, food safety practices, and dedication to sustainable practices make California the most important strawberry production area in the world. California grew 16,303 hectares of strawberries in 2023 in three growing districts located along California’s Central Coast: Oxnard, Santa Maria and Watsonville-Salinas. Transplants are grown on approximately 1,627 ha at high and low elevation nurseries located up to a thousand km from fruit growing districts. Almost all fruit production is in open fields with a small amount under high plastic tunnels either in soil or on tabletops. Nursery production is almost all in open fields. In 2022, there were 13 public cultivars grown with the most popular being Monterey, Portola, Fronteras, Cabrillo, and San Andreas grown on 27%, 13%, 11%, 4% and 2% of the planted acres, respectively. All proprietary cultivars combined made up 39% of the planted acres. Fresh market makes up 81% of the harvested fruit with 19% destined for processing into concentrates, individually quick frozen, purees and juice. Most fruit are sold domestically (87%) while 13% is exported, the majority to Canada (62%) and Mexico (25%). The most important diseases are Macrophomina root rot, Fusarium wilt, Verticillium wilt, Phytophthora root rot, Botrytis fruit rot and powdery mildew. The most important arthropod pests are twospotted spider mite and Lygus bug. Current challenges include increased regulation on water quality, fumigants, pesticides, labor and increased urbanization.
... The strawberry industry relies primarily on clonal propagation of daughter plants or crown division to generate new plant material for fruit producers. Compared with field production, using controlled environments to produce strawberry daughter plants offers the advantages of reduced disease pressure (especially of soil-borne pathogens), better control of the growing environment, and year-round availability of actively growing starting plant material (Baggio et al. 2021a(Baggio et al. , 2021bHern andez-Mart ınez et al. 2023;Xu and Hern andez 2020). These benefits have led to research aimed at improving strategies for growing mother plants in controlled environments to increase the runner number, daughter plant number, and daughter plant quality (Shi et al. 2021;Xu and Hern andez 2020). ...
Hydroponic growing systems are advantageous for nutrient studies in which root data are important because they alleviate the laborious and time-consuming task of washing roots to remove soilless substrate particulates from them. However, the growing system should be optimized for the crop of interest. Our overall objective was to develop a protocol for hydroponic strawberry ( Fragaria × ananassa ) production that provided growth equal to or better than soilless substrate. Plants were initially grown in perlite, sand, deep water culture (DWC), or a peat-based soilless substrate. Aboveground plant growth in DWC was similar to that of plants grown in the peat-based substrate and required minimal effort to harvest the entire root system. However, the pH of the DWC nutrient solution decreased to 4.0 ± 0.1 (mean ± SE ) when plants were provided a modified strawberry (Yamazaki) nutrient solution with a ratio of nitrate (NO 3 ⁻ ) to ammonium (NH 4 ⁺ ) of 80:20. As a result, a subsequent trial was conducted to evaluate the buffering capacity of nutrient solutions with NO 3 ⁻ to NH 4 ⁺ ratios of 0:100, 20:80, 50:50, 60:40, 80:20, or 100:0, with the addition of potassium bicarbonate (KHCO 3 ). Up to 2.6 mM KHCO 3 did not provide adequate buffering in nutrient solutions containing NH 4 ⁺ (0:100 to 80:20 treatments), and nutrient solution pH decreased by ∼1.5 units every 2 to 3 days. The 100% NO 3 ⁻ nutrient solution, however, maintained a stable pH of 5.9 ± 0.1 when buffered with 0.8 mM KHCO 3 . Finally, 2(N-Morpholino)ethanesulfonic acid (MES) was evaluated as a potential buffering agent for DWC strawberry production. Plants were grown in a nutrient solution with a 60:40 ratio of NO 3 ⁻ :NH 4 ⁺ . The buffering capacity of the nutrient solution increased as the MES concentration supplied increased from 1 to 5 mM, and the 5 mM MES treatment maintained a pH of 5.6 ± 0.2. In summary, strawberry plants can successfully be grown hydroponically in DWC, provided that nutrient solution pH is adequately managed. The addition of MES buffer provided better pH stability than KHCO 3 .
... They have been identified as plant pathogens (Tsai et al. 2018(Tsai et al. , 2021Fiorenza et al. 2022;Xiong et al. 2022;Zhang et al. 2022;Sun et al. 2023), human pathogens (Monden et al. 2013), saprobes Sun et al. 2023) and endophytes (Maharachchikumbura et al. 2011;Sun et al. 2023). Neopestalotiopsis species have recently been identified as a group of emerging plant pathogens, causing severe diseases on economically important crops and fruits, such as strawberry (Baggio et al. 2021), guava (Solarte et al. 2018;Bhogal et al. 2022), grape (Huanaluek et al. 2021), mangosteen (Huanaluek et al. 2021), avocado (Fiorenza et al. 2022), blueberry (Santos et al. 2022), jabuticaba ) and persimmon (Qin et al. 2023). Apart from that, many pestalotiopsis-like fungal species have been identified as promising in terms of producing novel biologically active compounds (Xie et al. 2014;Deshmukh et al. 2017). ...
Pestalotiopsis sensu lato, commonly referred to as pestalotiopsis-like fungi, exhibit a broad distribution and are frequently found as endophytes, saprobes and pathogens across various plant hosts. The taxa within pestalotiopsis-like fungi are classified into three genera viz. Pestalotiopsis, Pseudopestalotiopsis and Neopestalotiopsis, based on the conidial colour of their median cells and multi-locus molecular phylogenies. In the course of a biodiversity investigation focusing on pestalotiopsis-like fungi, a total of 12 fungal strains were identified. These strains were found to be associated with stromata of Beauveria, Ophiocordyceps and Tolypocladium in various regions of Taiwan from 2018 to 2021. These strains were evaluated morphologically and multi-locus phylogenetic analyses of the ITS (internal transcribed spacer), tef1-α (translation elongation factor 1-α) and tub2 (beta-tubulin) gene regions were conducted for genotyping. The results revealed seven well-classified taxa and one tentative clade in Pestalotiopsis and Neopestalotiopsis. One novel species, Pestalotiopsis manyueyuanani and four new records, N. camelliae-oleiferae, N. haikouensis, P. chamaeropis and P. hispanica, were reported for the first time in Taiwan. In addition, P. formosana and an unclassified strain of Neopestalotiopsis were identified, based on similarities of phylogeny and morphology. However, the data obtained in the present study suggest that the currently recommended loci for species delimitation of pestalotiopsis-like fungi do not deliver reliable or adequate resolution of tree topologies. The in-vitro mycelial growth rates of selected strains from these taxa had an optimum temperature of 25 °C, but growth ceased at 5 °C and 35 °C, while all the strains grew faster under alkaline than acidic or neutral pH conditions. This study provides the first assessment of pestalotiopsis-like fungi, associated with entomopathogenic taxa.
... La caracterización morfológica y la identificación filogenética son aproximaciones cercanas respecto a la identificación de hongos fitopatógenos como Neopestalotiopsis [7] (Figura 3). Las enfermedades causadas por hongos fitopatógenos se han vuelto cada vez más graves, como el reciente brote de Neopestalotiopsis en fresas en Florida [8]. Pestalotiopsis y géneros relacionados tales como Neopestalotiopsis y Pseudopestalotiopsis, infectan una amplia gama de hospedantes y actualmente causan problemas en todo el mundo. ...
La producción de berries a nivel internacional es de gran importancia por su rentabilidad económica. En el año 2020, se obtuvo una producción de 555,514 ton con un valor económico de $10,720,443.96. Los principales Estados productores de fresa son Michoacán, Baja California y Guanajuato. A nivel mundial, se cultiva principalmente en China, Estados Unidos de América, España y México, seguidos de Turquía, Egipto, República de Corea, Japón, Polonia y Rusia. La fresa sirve como fuente de nutrientes esenciales y fitoquímicos tales como antocianinas y elagitaninos. Actualmente, existe una plaga que acecha a los cultivos de frutillas, entre ellos la fresa. Esta enfermedad de la plaga se conoce como “secadera” ocasionada por el hongo Neopestalotiopsis. En el presente trabajo se menciona la importancia de la fresa y la plaga del hongo que ocasiona pérdidas del 100%.
... These characteristics were similar to those observed in the previous studies observing LFD in other plants, such as LFD in Royal Palm and Erythropalum scandens (Yang et al. 2021;Ismail et al. 2022). Temperature affected the mycelial growth of the Pestalotiopsis, with optimum growth ranging from 24 to 28ºC (Keith et al. 2006;Baggio et al. 2021) and failed to grow at temperatures below 20ºC or above 30ºC (Fovo et al. 2017;Bhuiyan et al. 2021;Li et al. 2021). The conidial characters in this study are similar to those reported by Febbiyanti and Fairuzah (2019) but showed that the conidia have a more significant, more extensive conidial morphology than those reported by Kusdiana et al. (2020). ...
Darojat MR, Ardie SW, Oktavia F, Sudarsono. 2023. New leaf fall disease in rubber-pathogen characterization and rubber clone resistance evaluation using detached leaf assay. Biodiversitas 24: 1935-1945. Leaf fall disease (LFD) has become a significant issue for rubber plantations worldwide. Over the last four years, a newly emerging LFD has posed an alarming problem in natural rubber production. The most efficient way to control LFD is to use resistance rubber clones. Therefore, this study aims to characterize the pathogen causing the newly emerging LFD and evaluate rubber clone resistance to the pathogen using detached-leaf assay. The fungal pathogens were isolated from 32 F1 progenies of PB 260 x SP 217 crosses, and the fungi were characterized and identified based on their morphological and molecular characteristics. The results showed that the isolated pathogen causing LFD was Neopestalotiopsis sp. Although they were all pathogenic, the arrays of isolated fungi exhibited various degrees of virulence, and P-212 was the most virulent fungal isolate. The resistance evaluation showed that rubber clones, isolates, and rubber clones by isolate interactions had a significant (p<0.05) effect on the lesion-symptom diameters. Based on the lesion diameter responses, the IRR 112 and RRIC 100 rubber clones were resistant to Pestalotiopsis sp. since they only showed less than 10 mm lesion diameters. The IRR 39 and PB 260 rubber clones were susceptible and showed more than 20 mm lesion diameters. The detached-leaf assay can easily screen rubber clones' responses to the fungi causing LFD. The resistance evaluation results can assist future rubber breeding strategies for the newly emerged LFD-resistant characters.
... This is of immediate concern with respect to a devastating and newly emerged pathogen, Neopestalotiopsis spp. causing leaf spot and fruit rot (Baggio et al. 2021a), which has devasted fruit production in Florida and many eastern regions of the United States, and may spread globally through nursery plants. Used judiciously, captan application is an economical, effective, and reliable tool for strawberry growers to manage BFR and AFR, increase marketable yield, and reduce the incidence of culls. ...
Botrytis Fruit Rot (BFR) and Anthracnose Fruit Rot (AFR) are diseases of concern to strawberry growers. Both diseases are managed mainly by fungicide applications from the nursery (plant production) to the end of the growing season (fruit production). In Florida, captan is the main broad-spectrum fungicide used to control BFR and AFR. It has been tested in many trials over the years in various programs in alternation with single-site fungicides or weekly applications. Due to its broad-spectrum activity, captan is a pivotal tool in fungicide resistance management, as resistance to several fungicides have been reported in populations causing BFR and AFR. Our objective was to determine the effectiveness and profitability of applications of captan for controlling BFR and AFR based on a univariate meta-analysis considering data from 25 field trials conducted from 2005 to 2021 in Florida. Captan applications significantly improved marketable yields and reduced BFR and AFR incidence during late and total season periods. Marketable yields were significantly improved even when the effect on disease control was not significant. Our results indicate 100% probability that weekly captan applications would return the investment during low, medium, and high strawberry pricing regimes, although the magnitude of the return will depend on strawberry market prices. However, the probabilities of reducing BFR, AFR, and culls were lower. Results from our meta-analysis demonstrate the value of captan as an important tool for strawberry growers. Captan applications, besides effectively controlling BFR and AFR and improving marketable yields, will result in investment returns at any strawberry price level.
... Conversely, an emerging disease, primarily affecting fruit and foliage, but also known to stunt plants and cause crown and root symptoms in Florida strawberry was associated with nursery transplants. This disease, caused by Neopestalotiopsis spp., now poses a significant threat throughout much of the state's commercial strawberry production region (Baggio et al. 2021;Fig. 11). ...
Soil disinfestation is a method of pest control that has been practiced since the late 1800s, with the application of steam. This was followed by the development of chloropicrin for soil fumigation in the 1920s. In the 1950s, the introduction of methyl bromide as a soil fumigant changed numerous specialty crop agricultural production systems forever. This broad-spectrum fumigant provided pest control for major vegetable and berry fruit crops, particularly in California and Florida for more than 50 years. It allowed growers to dramatically increase yields, but its loss due to its negative impact on the ozone layer left producers without a stand-alone tool to control existing and emerging pests. In the last 20 years, focused research has resulted in the optimization of application strategies for existing alternative chemical fumigants, the development of new chemicals, and extraordinary progress in utilizing non-chemical soil disinfestation methods and biologically based systems for pest control.
... The pathogenicity test was conducted on twoyear-old P. guajava healthy plants, grown in earthen pots under natural conditions. Leaves of 120 plants were inoculated by spraying the conidial suspension of N. guajavicola (1 × 10 6 spores/ml) (Baggio et al., 2021 ...
... Due to the emergence of the novel fungal pathogen Neopestalotiopsis spp. in strawberry during both field seasons, some plots may have been unevenly affected by the presence of the disease. 29 Affected plants may have been unattractive to thrips and mites leading to abandonment of the plants on their own. However, spinetoram application consistently reduced the number of thrips, improved fruit yield, and reduced the number of thrips-damaged fruit. ...
BACKGROUND
Major pests of Florida strawberry (two‐spotted spider mites, Tetranychus urticae Koch; chilli thrips, Scirtothrips dorsalis Hood; and western flower thrips, Frankliniella occidentalis Pergande) are difficult to manage using only conventional chemical control. Previous research has demonstrated high mortality of T. urticae eggs after exposure to ultraviolet B (UV‐B) light. Additionally, ultraviolet C (UV‐C) light has been shown to suppress powdery mildew in strawberry at doses between 85 and 200 J m⁻² with no damage to the plant. Therefore, UV‐C may also have the potential to be used as an integrated pest management tool for arthropod pests of strawberry. The objectives of this study were to: (i) determine the effect of UV‐C on T. urticae, S. dorsalis, and F. occidentalis natural populations in open‐field strawberry; and (ii) determine the effect of UV‐C on T. urticae egg hatch after application in open‐field strawberry. Field studies were conducted during the 2019–2020 and 2020–2021 strawberry seasons in Florida, USA. Four treatments were compared: (i) foliar application of spinetoram in response to natural pest pressure; (ii) application of UV‐C 200 J m⁻² twice a week; (iii) application of UV‐C 350 J m⁻² twice a week; and (iv) a non‐treated control.
RESULTS
In the field trials, suppression of T. urticae was observed at 350 J m⁻² in 2020–2021. In the other field trials, no effect was observed due to low natural infestations. No negative impact on yield was observed from UV‐C applications.
CONCLUSION
UV‐C shows promise as a component of an integrated pest management program for T. urticae in strawberry. © 2022 Society of Chemical Industry.
... Although the relative abundance of Colletotrichum was higher in the ozonated water treatment group and the pharmaceutical treatment group than in the CK group, overall, the relative abundance of Colletotrichum was not high in either group, and the strawberry's own defense mechanisms resisted the invasion of these pathogens, and anthracnose had not yet occurred. The pathogenic fungi of strawberry root rot are more complex, and Pestalotiopsis and Colletotrichum can cause strawberry root rot (Salinas et al., 2020;Baggio et al., 2021). Botrytis cinerea and P. aphanis are the causal agents of gray mold and powdery mildew, respectively, in strawberry (Kamaruzzaman et al., 2018;Feng et al., 2020). ...
Phyllosphere microorganisms are closely linked to plant health. This study investigated the effect of ozonated water, mancozeb, and thiophanate-methyl on phyllosphere microorganisms in strawberry plants of the “Hongyan” variety. Sequencing analysis of the phyllosphere bacterial and fungal communities was performed using 16S rRNA gene fragment and ITS1 region high-throughput sequencing after spraying ozonated water, mancozeb, thiophanate-methyl, and clear water. Proteobacteria, Actinobacteria, and Firmicutes were the dominant bacterial phyla in strawberry. The relative abundance of Proteobacteria (82.71%) was higher in the ozonated water treatment group than in the other treatment groups, while the relative abundance of Actinobacteria (9.38%) was lower than in the other treatment groups. The strawberry phyllosphere fungal communities were mainly found in the phyla Basidiomycota and Ascomycota. The relative abundance of Basidiomycota was highest in the ozonated water treatment group (81.13%), followed by the mancozeb treatment group (76.01%), while the CK group only had an abundance of 43.38%. The relative abundance of Ascomycota was lowest in the ozonated water treatment group (17.98%), 23.12% in the mancozeb treatment group, 43.39% in the thiophanate-methyl treatment group, and 55.47% in the CK group. Pseudomonas, Halomonas, and Nesterenkonia were the dominant bacterial genera on strawberry surfaces, while Moesziomyces, Aspergillus, and Dirkmeia were the dominant fungal genera. Ozonated water was able to significantly increase the richness of bacteria and fungi and decrease fungal diversity. However, bacterial diversity was not significantly altered. Ozonated water effectively reduced the relative abundance of harmful fungi, such as Aspergillus, and Penicillium, and enriched beneficial bacteria, such as Pseudomonas and Actinomycetospora, more effectively than mancozeb and thiophanate-methyl. The results of the study show that ozonated water has potential as a biocide and may be able to replace traditional agents in the future to reduce environmental pollution.
... Under favorable conditions, the pathogen spreads very rapidly. Primary source of infection is infected planting material (Baggio et al. 2021). ...
Highbush blueberry (Vaccinium corymbosum L.) is becoming an important berry fruit in Serbian agriculture, and the areas under blueberries are rapidly expanding. Since 2011, blueberry bushes with the symptoms of twig dieback and shoot canker were noticed in four important producing regions in the country. To identify the causal agent, 40 symptomatic bushes from four districts were sampled and analyzed. Based on morphological characteristics, all analyzed isolates were preliminary identified as Neopestalotiopsis clavispora. Selected isolates were further characterized by sequence analysis of the rDNA internal transcribed spacer region, translation elongation factor 1-alpha, and β-tubulin (TUB). Sequence and phylogenetic analysis confirmed that all isolates belong to N. clavispora. The pathogenicity tests on blueberry ‘Elliot’ confirmed that tested isolates were pathogenic. The detection of N. clavispora, a causal agent of twig dieback on blueberries in Serbia, is the first report of this pathogen in the country.
... fragariae and Neopestalotiopsis spp. (Baggio et al. 2021), which, hence, could make this method an even more valuable and attractive tool for routine use in a plant diagnostic clinic. ...
Rapid and accurate disease diagnosis is a prerequisite for an effective disease management program in strawberry production. In Florida, Colletotrichum spp., Phytophthora spp, and Macrophomina phaseolina are the primary microorganisms causing strawberry crown rot. Even though the diseases can be caused by different pathogens, symptoms are indistinguishable and equally devastating. To timely inform strawberry growers of diagnostic results for effective deployment of chemical control practices, we developed a multiplex high-resolution melting (HRM) assay to rapidly and accurately detect the above-mentioned pathogens. The multiplex HRM assays using three pre-designed primer pairs showed high specificity for individual species by generating specific melting peaks without cross-reaction between primers or with other common strawberry pathogens. The amplification limit of the assay was 1 pg of Colletotrichum and Phytophthora and 100 pg of M. phaseolina DNA per 10 μl reaction. However, the presence of different melting peaks was observed in mixed DNA samples and was concentration- and target DNA-dependent. A crude DNA extraction protocol was developed to allow high-throughput screening by minimizing the inhibitory effects. Moreover, we applied the HRM assay to 522 plant samples and found high correlations between conventional pathogen isolation and HRM and between singleplex and multiplex assays. Altogether, this multiplex HRM assay is specific, cost-effective, and reliable for the timely detection of strawberry crown rot pathogens.
The annual production of strawberry has increased by one million tonnes in the US and 8.4 million tonnes worldwide since 1960. Here we show that the US expansion was driven by genetic gains from Green Revolution breeding and production advances that increased yields by 2,755%. Using a California population with a century-long breeding history and phenotypes of hybrids observed in coastal California environments, we estimate that breeding has increased fruit yields by 2,974-6,636%, counts by 1,454-3,940%, weights by 228-504%, and firmness by 239-769%. Using genomic prediction approaches, we pinpoint the origin of the Green Revolution to the early 1950s and uncover significant increases in additive genetic variation caused by transgressive segregation and phenotypic diversification. Lastly, we show that the most consequential Green Revolution breeding breakthrough was the introduction of photoperiod-insensitive, PERPETUAL FLOWERING hybrids in the 1970s that doubled yields and drove the dramatic expansion of strawberry production in California.
Devastating disease outbreaks with leaf spot symptoms and fruit rot caused by a fungus identified as a cryptic species of Neopestalotiopsis have generated concern in the Florida strawberry industry. Some Neopestalotiopsis species are saprobes in soil and plant debris, but the ability of the new Neopestalotiopsis sp. to survive in strawberry debris was unknown. Therefore, the objectives of this study were to develop a semi-selective medium for isolation of Neopestalotiopsis spp. and to evaluate Neopestalotiopsis sp. survival in soil and strawberry plant debris over summer in Florida. The Neopestalotiopsis semi-selective medium (NSM) inhibited growth of most fungal species pathogenic to strawberry in Florida, except Neopestalotiopsis rosae, Neopestalotiopsis sp., and Phomopsis obscurans, in addition to Penicillium spp., which is non-pathogenic. However, Neopestalotiopsis species, P. obscurans, and Penicillium spp. could be distinguished in the medium through morphological characteristics. Soil samples arbitrarily collected from six commercial fields towards the end of the season (May), and before (July) and after (September) pre-plant soil fumigation in the following season were processed with NSM and Neopestalotiopsis spp. populations were calculated as CFU/g soil. CFU ranged from 48.3 to 2410.8 at the end of the season and from 1.7 to 630.8 before soil fumigation, but Neopestalotiopsis spp. was not recovered after soil treatment. However, 1.7 to 25 CFU were obtained from the non-treated areas in the row middles. Neopestalotiopsis sp. survival was also evaluated on diseased strawberry plants (leaves and crowns) exposed to the environment for 17 months. On leaves, Neopestalotiopsis spp. was recovered for 6 months before leaf decomposition, whereas the number of CFU on crowns declined monthly but colonies were still recovered during the final evaluation months. HRM analysis confirmed most of the colonies from soil and crowns were the new Neopestalotiopsis sp. Our results show Neopestalotiopsis sp. can survive in strawberry debris under Florida summer conditions and may serve as a source of inoculum for the subsequent season.
Synsepalum dulcificum is one of the well- known edible and ornamental plants with
its unique flavour changing function of sour taste. In 2021, a new leaf spot disease
was observed on S. dulcificum during a field survey in Guangzhou City, Guangdong
Province, China. The pathogen was isolated and identified using morphological examination and phylogeny based on multi- gene sequences analysis of the nuclear ribosomal internal transcribed spacer (ITS), translation elongation factor 1- alpha (TEF), and beta- tubulin (TUB) genes. The isolates were identified as Neopestalotiopsis iberica. The pathogenicity of N. iberica on S. dulcificum was validated fulfilling Koch's postulates, causing leaf spots disease. To our knowledge, this represents the first report of leaf spot disease on S. dulcificum caused by N. iberica worldwide.
Fruit and vegetable crops are important sources of nutrition and income globally. Producing these high value crops requires significant investment of often scarce resources, and therefore the risks associated with climate change and accompanying disease pressures are especially important. Climate change influences the occurrence and pressure of plant diseases, enabling new pathogens to emerge and old enemies to re-emerge. Specific environmental changes attributed to climate change, particularly temperature fluctuations and intense rainfall events, greatly alter fruit and vegetable disease incidence and severity. In turn, fruit and vegetable microbiomes, and subsequently overall plant health, are also affected by climate change. Changing disease pressures cause growers and researchers to reassess disease management and climate change adaptation strategies. Approaches such as climate smart IPM, smart sprayer technology, protected culture cultivation, advanced diagnostics, and new soilborne disease management strategies are providing new tools for specialty crops growers. Researchers and educators need to work closely with growers to establish fruit and vegetable production systems that are resilient and responsive to changing climates. This review explores the effects of climate change on specialty food crops, pathogens, insect vectors, and pathosystems, as well as adaptations needed to insure optimal plant health and environmental and economic sustainability.
Rosa ‐associated fungi are rich in species and diverse in function, with many potential new taxa. However, only a few publications have reported Rosa ‐associated endophytic fungi using a combination of molecular systematics and morphology. Rosa roxburghii is an economically important source of medicine and food. We reported earlier the diversity and antimicrobial activities of the fungal endophytes of this plant, but further confirmation is needed for possible new taxa. In this study, we describe 10 novel species and two new host records isolated from healthy tissues of R. roxburghii while identifying three new combinations, all belonging to the Sordariomycetes. The new species described herein include Chaetomium rosifloricola , Chaetomium rosiradicicola , Chrysofolia fructicola , Coniella cili , Hypoxylon florendophyticum , Hypoxylon rosicaulendophyticum , Ilyonectria radicigena , Neopestalotiopsis caulicola , Neopestalotiopsis fructicola , and Seimatosporium endophyticum . Monochaetia bulbophylli , Neopestalotiopsis anacardii , and Neopestalotiopsis termitarii were introduced as new combinations, while Coniella quercicola and Hypoxylon pulicicidum were described as new host records on R. roxburghii . All the taxa have been described, illustrated, and phylogenetically analyzed, in addition to the lifestyles of each genus. Altogether, the results of this study have important implications for identifying novel endophytic fungi and developing potential applications of these fungi in the future.
Identifying the precise pathogens responsible for specific plant diseases is imperative for implementing targeted and efficient interventions and mitigating their spread. Dieback and shoot blight significantly diminish the lifespan and productivity of blueberries, yet the causative agents remain largely unidentified. To determine the identity and prevalence of the causal agents of branch dieback and shoot blight, we conducted multi-year and multi-site sampling of diseased highbush blueberries (Vaccinium corymbosum cv. Duke) in Serbia. Sixty-nine monosporic isolates were collected and characterized based on morphological, physiological features and multi-locus phylogenetic analysis of internal transcribed spacer (ITS), β-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1) sequence data. Five species were identified as causal agents: Diaporthe eres (36 isolates), D. foeniculina (3 isolates), Neopestalotiopsis vaccinii (9 isolates), N. rosae (6 isolates) and Neofusicoccum parvum (15 isolates). The results of the pathogenicity tests performed with the 23 representative isolates confirmed the role of these species as primary pathogens in causing dieback and shoot blight of blueberry, with N. parvum being the most aggressive and D. eres the least. Our study underscores the diversity of genera and species of ascomycetes capable of causing blueberry dieback and shoot blight. Furthermore, our findings indicate that the agents responsible for the disease in Serbia differ from those identified in other regions worldwide.
Pestalotiopsis-like species are necrotrophic fungi, which infect many annual and perennial crops, including agricultural, horticultural, and plantation crops, in postharvest and under field conditions worldwide. They cause multiple diseases on crops, which results in severe yield loss. At present, Pestalotiopsis-like species cause gray blight on tea, which is a widely prevalent disease in major tea-growing countries and rapidly spreading in other teagrowing countries of minor importance due to climate change. The global increase in disease incidence and severity and the emergence of new virulent isolates have prompted research on the evolution of pathogenic determinants in these fungal species. This review synthesizes the epidemiology, molecular and genetic studies of the gray blight pathogen with particular reference to tea crop and the approaches to mitigate it. Further, the adaptation of Pestalotiopsis-like species on other crops and their management strategies are also discussed along with potential areas for future research.
Goal. To identify fungi associated with the tissues of strawberry plants and evaluate the frequency of their isolation at different stages of plant growth. Methods. The research was conducted in the Right Bank Forest Steppe of Ukraine (Cherkasy Region) in 2021—2023, cv. ‘Clery’. Plant samples were taken in the phases of rosette formation (April), budding-flowering (May), fruit ripening (June) and in the post-harvest period (July-August). The analysis was carried out in laboratory conditions using macroscopic and biological methods. Results. During the research period, fungi belonging to 27 genera were isolated from strawberry tissues. In general, they can be divided into three groups: pathogens of leaves, which is the most numerous, berries, roots and crown. Representatives of the genera Alternaria, which were isolated from 57—100% of samples, and Fusarium (40—88%) occurred most often. Less often, but also during the entire growing season, Penicillium spp. (36—69%), Podosphaera aphanis (11—80%), Botrytis cinerea (27—67%), Paraphomopsis obscurans (24—77%) were isolated. Among the leaf diseases powdery mildew was recorded during all growing season, with a maximum in the fruit ripening phase. White leaf spot was observed starting from the budding-flowering phase, and leaf scorch was found from the fruit ripening phase. Gray rot was the most common fruit disease. The root system was more often affected by Fusarium spp. Conclusions. The obtained results demonstrate that during the entire growing season fungi of the genera Alternaria, Fusarium, Penicillium, as well as Podosphaera aphanis, Botrytis cinerea and Paraphomopsis obscurans were most often isolated from strawberry tissues. R.grevilleana was isolated starting from the budding-flowering phase, and D. earlianum from the fruit ripening phase. Species from the genera Podosphaera, Ramularia, Diplocarpon, Colletotrichum, Pestalotiopsis, Verticillium, Sclerotinia, Gnomoniopsis, Rhizoctonia, Coniella, Rhizopus, Septoria, Cylindrocarpon require constant control of their spread. Under favorable conditions for their development, they can cause damage to plants and a significant lack of harvest. The seasonal monitoring of fungi on strawberry provides a means for establishing the optimal periods for their control and developing effective disease protection system.
Strawberry is a popular fruit with valuable nutrition and an attractive fragrance, but its production and propagation are limited by various diseases, such as anthracnose and gray mold. For disease management, biological control measures are environmentally friendly and good alternatives to fungicides to avoid crop losses, reduce carbon emissions, and improve food safety. In this study, Paenibacillus polymyxa TP3, which originated from the strawberry phyllosphere, was shown to antagonize the anthracnose fungal pathogen Colletotrichum siamense and reduce leaf symptoms on strawberry plants. Several fusaricidin-corresponding mass spectra were detected in the confrontation assay of P. polymyxa TP3 and C. siamense by image mass spectrometry. The transcription of fusA and fusG in the fusaricidin biosynthesis gene cluster increased while P. polymyxa TP3 was cultured in the medium containing the culture filtrate of C. siamense, as detected by reverse transcriptase-polymerase chain reaction, indicating the involvement of fusaricidins in P. polymyxa TP3 antagonism of the anthracnose pathogen. Further disease control assays demonstrated the time frame and spatial mode of P. polymyxa TP3-induced systemic resistance of strawberry against C. siamense. The transcript level of the marker gene FaPDF1.2 of the jasmonic acid pathway was increased in strawberry leaves after drenching treatment with P. polymyxa TP3, and the callose deposition was enhanced by further flg22 treatment. In addition, P. polymyxa TP3 treatments of the mother strawberry plants reduced C. siamense infection in the daughter plants, which would be a potent feature for the application of P. polymyxa TP3 in the strawberry nursery and the field to reduce the disease impact, especially of anthracnose.
This study aims to generate a highly effective nano-bioformulation from essential oils (lemongrass oil, mustard oil, thyme oil, eucalyptus oil, chili oil, cinnamon oil, mint oil) for controlling N. rosae, a fungus responsible for causing leaf spot disease on strawberries in Vietnam. Firstly, the plant pathogen was isolated from diseased strawberry samples and identified using potato dextrose agar medium. After that, essential oil-encapsulated lipid nanoemulsions (EO-LNs) were prepared by a combining method of homogenization and sonication. Their binary and ternary combined formulations were also prepared by mixing with an equal volume ratio of each EO-LNs. In vitro antifungal activity of 7 EO-LNs and their combined formulations were evaluated using petri dish assay. The resultant findings were then used so that the most optimal EO-LN of all is found. Next, it was field-tested for its effectiveness regarding ex vitro disease-preventing and disease-treating. The test produced three results; first, it confirmed that the isolated fungus that causes leaf spots on strawberries was Neopestalotiopsis rosae. Second, in vitro results indicated that cinnamon oil-encapsulated LNs (CiLN) exhibited highest antifungal activity among 7 EO-LNs (single formulations) and even higher than that of the binary and ternary combined formulations, reached to 95.59% at 500-time dilution. Third, in the field tests, CiLN showed high effectiveness in strawberry’s disease-preventing and disease-treating at 100% and 90.42%, respectively. This is the first report mentioning EO-LNs formulation as a biofungicide for effectively controlling N. rosae causing leaf spot disease on strawberry plants without causing any negative effect on the plant growth.
In June 2020, a leaf spot caused by Neopestalotiopsis sp. in Indiana strawberry production was confirmed to be the same disease recently found in Florida strawberry production. Differences in cultivar susceptibility were observed in the field. Greenhouse trials using either bare-root or plug plants were conducted to explore possible differences in host resistance to the foliar phase of the disease caused by Neopestalotiopsis sp. Clear differences in cultivar susceptibility were observed. Florida Brilliance, Florida Sensation, Camino Real, and Florida Radiance were among the most susceptible cultivars evaluated in these trials. Several short-day cultivars including AC Valley Sunset, Darselect, Earliglow, Flavorfest, Galetta, Honeoye, Jewel and Sonata remained asymptomatic in the trials. Since the above-mentioned asymptomatic short-day cultivars are commonly used in matted row systems typical of Indiana strawberry production, future field trials with the matted row system may demonstrate that the foliar phase of the disease caused by Neopestalotiopsis sp. may be less common in Indiana than in other areas of the US.
Recently, the Florida strawberry industry faced unprecedented outbreaks of an emerging disease caused by the fungus Neopestalotipsis spp. Currently, there are no fungicides labeled to control this disease in the U.S. and the efficacy of single- and multi-site fungicides is unknown. Therefore, this study aimed to determine the in vitro sensitivity of Neopestalotiopsis spp. isolates to fungicides with different modes of action and to evaluate the efficacy of these products on detached fruit and in the field. In preliminary in vitro tests, 30 commercially available fungicides were screened using discriminatory doses. The effective concentration that inhibited mycelial growth by 50% (EC50) was determined for the most effective single-site fungicides. Four field experiments were conducted during the 2019-20, 2020-21, and 2021-22 seasons to determine product efficacy in managing the disease. The single-site fungicides fludioxonil, fluazinam, the sterol de-methylation inhibitors (DMIs), and the multi-sites captan, thiram, and chlorothalonil were the most effective in inhibiting pathogen growth and suppressing disease development. Conversely, products in FRAC groups 1 (MBC) and 7 (SDHI), except for benzovindiflupyr, were not effective against Neopestalotiopsis spp. Resistance to fungicides from FRAC group 11, e.g., azoxystrobin, was confirmed by the presence of the G143A mutation in the cytochrome b gene together with inoculation tests and field trials. Our results provide information to support or discourage the registration of fungicides to manage Neopestalotiopsis fruit rot and leaf spot in strawberry production. .
A new Neopestalotiopsis sp. was recently reported causing outbreak of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida, isolates of N. rosae, N. honoluluana, N. ellipsopora, N. saprophytica, N. samarangensis, and P. rhododendri, and isolates from South Carolina suspected to be the new Neopestalotiopsis sp were included in this study. This method is based on PCR amplification of a β-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice yielding fragments of 290 base pairs (bp) and 130 bp and 20 bp in size, whereas fragments from other species are only cut once yielding fragments of 420 bp and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry.
Since 2017, a new Neopestalotiopsis sp. has been reported causing severe Pestalotia leaf spot (PLS) outbreaks in Florida. The threat of this new species has forced several strawberry growers to destroy their fields early in the season. The need for a fast and accurate diagnosis of PLS came from growers’ concerns and the fact that PLS symptoms can be easily confused with leaf spots caused by other pathogens. Moreover, although N. rosae can be isolated from symptomatic tissues, it is not as aggressive as the new Neopestalotiopsis sp. in Florida. Therefore, this study aimed to develop a high-resolution melting (HRM) assay to accurately detect and differentiate Neopestalotiopsis spp. associated with strawberry and validate this assay for its practical application in a plant diagnostic clinic. Two sets of primers were designed based on the partial beta-tubulin gene (β -tub) and by cross-comparison of their HRM results. This molecular assay was able to differentiate Neopestalotiopsis spp. into three groups: the new Neopestalotiopsis sp. associated with PLS outbreaks, N. rosae, and other Neopestalotiopsis spp. represented by several other species within the genus. The HRM assay had a detection limit of 10 pg of genomic DNA or 10 conidia and was found to be efficient and accurate to be implemented in our plant diagnostic clinic, allowing for a quick turnaround of results.
[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 – GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~10 ⁴ spore/ml) four-week-old ‘Cabrillo’ strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~10 ⁴ spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.
Strawberry production in California represents over 38,000 acres with an annual farm value of $1.99 billion. Strawberry dieback was observed in February of 2021 in the Salinas Valley in central California. Disease symptoms included dead and dying ‘Maverick’ strawberry plants with necrotic lesions and black discoloration of the crown, root cortex, epidermis, and vascular tissues. Disease incidence was estimated to be 60% of a 20-acre field. The causal agent was isolated from five randomly selected symptomatic plants by surface disinfesting symptomatic crowns and roots in 1% sodium hypochlorite for 30 s, rinsed twice in sterile water for 30 s then placed on 3.7% potato dextrose agar (PDA) Petri dishes amended with 100 mg/L streptomycin, and then incubated for 7 days at 24°C under a 12-h photoperiod. Consistent white cottony fungal colonies were hyphal tip transferred to fresh PDA dishes and incubated as above for morphological and genetic comparisons. Black acervuli developed 7 to 9 days after incubation. Conidia were ellipsoidal, measuring 25 to 30 × 7.5 to 10 µm (average 26.8 × 9.2 µm, n = 30), with five cells. Apical and basal cells were hyaline, and the three median cells were versicolorous brown, with a single, straight, centric basal appendage and 3 to 4 flexuous apical appendages. Colony diameter averaged 90 mm in 7 days. Based on colony and conidial characters, the fungus was tentatively identified as a species of Neopestalotiopsis (Maharachchikumbura et al. 2014). Total genomic DNA was extracted from three axenic cultures using the Invitrogen Easy-DNA kit. Three genetic loci were PCR amplified and sequenced: internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factor 1-alpha (TEF) utilizing the primer pairs ITS1/ITS4, T1/Bt2a, and EF1-688F/EF1-1251R, respectively (White et al. 1990, O’Donnell and Cigelnik 1997, Glass and Donaldson 1995, and Alves et al. 2008). A BLASTn search of NCBI showed 99.6% identity (495/497 bases; OM942910-OM942912) with the type specimen of Neopestalotiopsis rosae CBS 101057 for the ITS locus. Both BT (765/765 bases, OM964802-OM964804) and TEF (475/475 bases; OM964799-OM964801) sequences were 100% identical to CBS 101057. Conidia of isolate PAR027 were scraped from the surface of 14-day-old PDA Petri dishes and inoculated (1 × 106 spores/mL: 2 mL/plant) to four apparently healthy strawberry transplant roots of the cultivar ‘Monterey’ in ‘sunshine mix’ potting soil. Two control plants were inoculated with sterile water. The experiment was conducted twice. Strawberry plants were maintained in a hoop house for four weeks, after which dieback and wilt symptoms resembled the symptoms observed in the field. Control plants remained asymptomatic and no pathogens were isolated. Fungal recovery from inoculated plants morphologically matched the original inoculum; thus, Koch’s postulates was satisfied. To our knowledge, this is the first report of N. rosae causing crown and root rot disease of strawberry in California. Previously, N. rosae has been reported to cause serious decline of strawberry plants in Florida and several countries (Baggio et al. 2021, Rebollar-Alviter et al. 2020, Wu et al. 2021, Sun et al. 2021). Correct identification of the causal agent provides a proper foundation to identify control strategies for this emerging disease, which has the potential to become a significant problem for strawberry growers in the Salinas Valley of California.
Aphelenchoides besseyi and A. pseudogoodeyi are foliar nematodes associated with commercial strawberry production in Florida, USA. The reproductive and feeding habits of these two nematode species were assessed on Florida isolates of the fungi Botrytis cinerea, Colletotrichum gloeosporioides, Macrophomina phaseolina, and Neopestalotiopsis spp. pathogenic to strawberry, and the non-pathogenic isolates of Fusarium oxysporum and Monilinia fructicola grown on PDA in petri dishes. Each culture was inoculated with six specimens with mix life stages of either A. besseyi and A. pseudogoodeyi and incubated at 24°C under axenic and non-axenic conditions 23 and 31 days after inoculation, respectively. Aphelenchoides besseyi reproduction rates were significantly higher on strawberry pathogenic isolates of B. cinerea, C. gloeosporioides, and Neopestalotiopsis rosae than on the non-pathogenic isolates of F. oxysporum and M. fructicola. In contrast, reproductive rates of A. pseudogoodeyi did not significantly vary across the fungi cultures. For both nematode species, Macrophomina phaseolina was a poor host because it did not produce mycelium on the media used. Our findings indicate that A. besseyi is more selective in its fungal-feeding preference than A. pseudogoodeyi. Additionally, A. pseudogoodeyi eggs and juveniles were significantly higher than adults. Yet, for A. besseyi, adult stages were more common. Fungi aid in the maintenance of soil-dwelling populations of these two nematode species. Removing fungi-infected strawberry plant residues is both a desirable and effective management practice to limit A. besseyi in central Florida commercial strawberry fields.
Pestalotiopsis and related genera, including Neopestalotiopsis and Pseudopestalotiopsis have damaged many plants for many decades; however, there is little available information about the fungi on tropical fruit in Thailand. This study isolated and characterized pestalotioid fungi on tropical fruit, investigated host specificity, and screened whether plant extracts could control the fungi. In total, 53 diseased fruit samples were sampled from eight types of fruit trees (jackfruit, rose apple, mangosteen, plum, snake fruit, rambutan, strawberry, and avocado). Based on morphological characteristics, 44 isolates were classified as belonging to pestalotioid taxa. Of these isolates, seven with distinct characteristics were selected for identification using molecular analysis, and six isolates were identified as Neopestalotiopsis and one as Pseudopestalotiopsis . In the cross-inoculation experiment, the isolates exhibited nonhost specificity and could infect at least two host plants. The isolates were used to screen for a potential biocontrol resource using six crude plant extracts (clove, ginger, lemongrass, mangosteen, roselle, and turmeric). All crude extracts except mangosteen could inhibit the growth of Neopestalotiopsis and Pseudopestalotiopsis . Application of crude plant extracts could be a potential treatment to control these diseases on tropical fruit.
Strawberry (Fragaria × ananassa) is widely cultivated in Liaoning Province, the most important strawberry production area in China. In November 2020, extensive brown spotting on the calyces and receptacles and dark brown necrotic lesions on the leaves of 'YanLi' strawberry plants were observed in the greenhouse, College of Horticulture, in Shenyang Agriculture University of Liaoning Province, China. The incidence ranged from 15% to 20% in the greenhouse. As the disease progressed, the spots extended to cover the entire receptacles and entire leaves. Fresh samples (5 mm2) were cut from the margin of healthy and infected tissues. The isolation and purification of methods were as described previously (Obregón et al. 2018). Fungal isolations are suborbicular with irregular edges. The colonies showed white aerial mycelium at the early stage and formed concentric circles after 5 days culture at 28°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 7~10 days culture. Acervulus was 162.4 µm in average diameter, n= 30. Conidia were spindle shape, five cells, four septa, 7.0 µm (average width) and 24.0 µm (average length), n=30. The apical and basal cells are lighter in color, most of them are hyaline, the middle three cells are darker in color, mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 30.8 µm in average length, and the basal cell has a small appendage, 9.0 µm in average length, n=30 (Fig. 1c-g). For fungal identification to species level, genomic DNA was extracted by the 2% CTAB method. Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. Purified PCR products were sequenced in single direction. BLASTn searches of GenBank, using the partial ITS (MW861730), β-tubulin (MW856302) and TEF-1α (MW861729) sequences of representative isolate NP102 as the queries, revealed 97%, 100% and 100% sequence identity, respectively, to Neopestalotiopsis clavispora strain WJ31 accessions MG729690, MG740736 and MG740759. The phylogenetic tree was constructed by MEGA X (Fig. 2) (Kumar et al. 2018). Pathogenicity assay was carried out by spraying a spore suspension (1 × 106 spores/ml) of 8-day-old culture of fungus on six healthy 'YanLi' strawberry plants. An additional six control plants were only sprayed with sterile distilled water, and the inoculated plants were maintained in a light incubator at 25 ± 1°C, 90% relative humidity, with a photoperiod of 12 h. Calyx and receptacle blight and leaf spot symptoms resembled those observed in the greenhouse on all inoculated tissues after 10 days (Fig. 1h and i), whereas calyces, receptacles and leaves on control plants remained asymptomatic. The experiment was repeated twice. Neopestalotiopsis clavispora was successfully reisolated from the infected tissues, fulfilling Koch's postulates. Researchers in Spain (Chamorro et al. 2016) reported N. clavispora for the first time on strawberry. After two years, N. clavispora was found in Argentina (Obregón et al. 2018). The reports characterized it mainly as a root rot pathogen. In January 2021, Neopestalotiopsis spp. were reported that it is a pathogen of strawberry leaves and fruits in Florida strawberry fields (Juliana et al. 2021). Our studies indicated that isolate NP102 was more aggressive. In the warm and humid greenhouse, the disease struck suddenly at the early stages of strawberry fruiting. Unlike previous reports, symptoms were a calyx and receptacle blight as well as a leaf spot. This pathogen can cause serious economic losses to strawberry growers. To the best of our knowledge, this is the first report of calyx and receptacle blight by N. clavispora on strawberry in China.
Late-season bunch rots cause major losses in grape production every year in the Mid-Atlantic United States, but the causal agents are not well characterized. In this study, 265 fungal isolates were collected from rotten grapes from 2014 to 2020 and identified to the genus level according to ITS sequences. The most prevalent of the 15 genera were Botrytis, Colletotrichum, Aspergillus, Alternaria, Pestalotiopsis, and Neopestalotiopsis. Of these, isolates within three prevalent, yet understudied genera were identified to be Aspergillus uvarum, Alternaria alternata, and Neopestalotiopsis rosae. The pathogenicity of these three fungal species was evaluated in two field trials by artificially inoculating wounded and non-wounded grapes of four cultivars at the phenological stages of bloom, veraison, and pre-harvest. Upon ripening, fruit were weighed and assessed for severity of multiple diseases. On non-wounded fruit, A. uvarum caused significantly higher disease severity than the control in both seasons. On wounded fruit, each inocula caused significantly higher disease than the respective controls in the first season, but only A. uvarum and B. cinerea caused this in the second season. Also, wounding was found to have a detrimental effect on cluster weight, which was significantly influenced by inoculation timing and cultivar. Lastly, A. uvarum and N. rosae were tested for sensitivity to azoxystrobin, boscalid, and difenoconazole. The A. uvarum isolates were found to be more sensitive to boscalid and difenoconazole in general, with varying sensitivity to azoxystrobin. N. rosae isolates were resistant to boscalid and azoxystrobin but displayed much higher sensitivity to difenoconazole. Evidence from the isolate collection and field trials demonstrates that A. uvarum could be a significant pathogen of wine grapes in the Mid-Atlantic. Results from this study will be useful for the identification and management of the understudied Alternaria, Aspergillus, and Neopestalotiopsis fruit rots of wine grapes.
Pestalotiopsis-like fungi cause diseases on many different species of plants worldwide, including strawberry. The pathogen is not necessarily new to strawberry and was first reported causing fruit rot in Florida and Israel in the 1970s. However, during the 2018–19 and 2019–20 strawberry seasons, severe and unprecedented outbreaks were reported in Florida, characterized by symptoms on nearly all plant parts including roots, crowns, petioles, fruit, and leaves. This new 4-page publication of the UF/IFAS Plant Pathology Department describes the disease and its causal agent and briefly covers what is known about disease development and control practices. This document will provide valuable information to strawberry nursery and fruit production growers. Written by Juliana S. Baggio and Natalia A. Peres.https://edis.ifas.ufl.edu/pp357
This publication describes the disease Pestalotia leaf spot and fruit ort and its causal agent and briefly covers what is known about disease development and control practices. This document will provide valuable information to strawberry nursery and fruit production growers.
Strawberry (Fragaria x ananassa Duch.) is a widely grown vegetable crop in Egypt. During a routine survey, wilt and root rot symptoms were observed on strawberry plants cv. Festival. Samples were collected and isolation was done on potato dextrose agar (PDA) medium. The frequently isolated fungi were Pestalotiopsis spp. along with other associated fungi. Koch’s postulates were fulfilled against two strawberry cultivars Fortuna and Festival under greenhouse conditions. Pestalotiopsis fungal isolates were identified by amplification and sequencing of ITS, TEF-1α and β-tubulin gene regions. The BLASTn search of GenBank database revealed that all obtained sequences exhibited similarity of 99–100% to Neopestalotiopsis rosae. Four fungicides were evaluated against N. rosae through in vitro and in vivo trials. In vitro trials revealed that fungicides Thiram and Hymexazole inhibited the mycelial growth of N. rosae at concentrations 1000 and 1250 ppm, respectively. In vivo trials showed that Thiram reduced significantly disease severity of crown and root rot on both cultivars
Species of Sporocadaceae are endophytic, plant pathogenic or saprobic, and associated with a wide range of host plants. Recent molecular studies that have attempted to address familial and generic boundaries of fungi belonging to Sporocadaceae were based on a limited number of samples and DNA loci. The taxonomy of this group of fungi is therefore still not fully resolved. The aim of the present study is to provide a natural classification for the Sporocadaceae based on multi-locus phylogenetic analyses, using LSU, ITS, tef-1α tub2 and rpb2 loci, in combination with morphological data. A total of 30 well-supported monophyletic clades in Sporocadaceae are recognised, representing 23 known and seven new genera. Typifications are proposed for the type species of five genera (Diploceras, Discosia, Monochaetia, Sporocadus and Truncatella) to stabilise the application of these names. Furthermore, Neotruncatella and Dyrithiopsis are synonymised under Hymenopleella, and the generic circumscriptions of Diploceras, Disaeta, Hymenopleella, Monochaetia, Morinia, Pseudopestalotiopsis, Sarcostroma, Seimatosporium, Synnemapestaloides and Truncatella are emended. A total of 51 new species, one nomina nova and 15 combinations are introduced.
Argentina cultivates ≈1,300 ha of strawberries (Fragaria × ananassa Duch.) and produces ≈45,500 t year-round. Since August 2013, severe damage has been observed on strawberry plants from commercial production fields, in the northern provinces of Corrientes (Bella Vista) and Tucuman (Famailla), Argentina. The main symptom was observed in leaves and consisted in reddish necrotic interveinal zones, with rust-colored areas in older leaves, starting from the margins and covering the whole leaf as the disease developed. This symptom was associated with necrotic areas and discoloration of roots and crown, resulting in plant death. According to colony and conidia morphology, the isolates were identified as Neopestalotiopsis clavispora.
This study was conducted to identify the crown rot disease in strawberry caused by an uncommon fungus.
However the study also focused to evaluate efficacy of plant extracts and chemical fungicides against this fungus. Results from isolating 200 samples of infected plant parts collected from 15 main strawberry cultivating areas in Rajshahi, Bangladesh. Based on morphological and cultural characteristics the Pestalotiopsis sp. was found as fungal pathogen against strawberry and Koch’s methodology confirmed it. Five different concentrations (50, 100, 250, 500 and 1000 ppm) of plant extracts (Neem, Tulsi, Bottlebrush, Arjun and Alovera) and commercial fungicides (Bavistin, Tilt, Hayconazole, Score, Rovral, Antracol, Dithane, Ridomil, Folicur, Cupravit, Secure, Thiovit and Sulcox) were used for efficacy study against Pestalotiopsis sp. in both in vitro and field condition. The percent inhibition of radial growth of the fungus was increased with the increasing concentrations of fungicide. Bavistin (1000 ppm) showed 100% inhibition of growth of this fungus in in vitro condition and in field condition. Bavistin treated plants showed only 21.5% disease incidence, where control was 85%. Number of fruits/plant was 10.88±0.23, fruit weight/plant was 148.56±0.81 (g) whereas in control these were 5.33±0.23 and 63.88±0.26 respectively. This is the first report on the association of Pestalotiopsis sp. causing crown rot disease of strawberries in Bangladesh.
Strawberry transplants produced in nurseries across Canada, northern United States, and California are shipped annually to other strawberry-growing regions, including Florida. Botrytis cinerea, the causal agent of gray mold, causes latent infections on transplants which are suggested as a potential source of primary inoculum in strawberry fields. In this study, we investigated the survival of B. cinerea isolates over the summer in Florida, the presence of B. cinerea in transplants from 14 nurseries from Canada and the United States in 2011, 2012, and 2013, and the sensitivity of nursery population to several botryticides. Botrytis cinerea was detected on dead strawberry plants sampled from commercial strawberry fields between March and June but not in July and August, suggesting that the fungus does not over-summer in strawberry fields in Florida. Nursery transplants surveyed in 2011, 2012, and 2013 showed B. cinerea incidences of 20 to 37, 20 to 83, and 2.5 to 92.5%, respectively. In total, 409 isolates were tested for sensitivity to pyraclostrobin, boscalid, pyrimethanil, fenhexamid, iprodione, penthiopyrad, fluopyram, and fludioxonil. Overall, respective resistance frequencies were 91.7, 79.3, 33.2, 20.7, 2.4, 0.2, 0.2, and 0.0%. A majority of isolates tested were resistant to either 3 or 4 fungicides simultaneously. These findings reinforce the need for an integrated approach between strawberry nurseries and production fields to improve gray mold management and mitigate future risks of resistance development in B. cinerea.
Strawberry (Fragaria x ananassa Duch.) is one of the most important crops in Huelva
(southwestern Spain) with 7,300 ha cultivated in 2012 yielding 275,300 tonnes (98% of the total
Spanish production). At the beginning of the 2013-14 season, a low incidence of mortality (~1%)
in strawberry plants cultivated at Huelva province was observed. Symptoms consisted in
discoloration or brown necrotic areas in crown and root (Fig. 1.A and 1.B, respectively). To
isolate the causal agent, crowns and roots were surface disinfected for 2 min in 1% sodium
hypochlorite, rinsed twice in sterile distilled water, air dried in a laminar flow-cabinet, and plated
on potato dextrose agar (PDA). Pure cultures of the fungus were grown on PDA at 25ºC with a
12-h photoperiod for 7-10 days. Fungal colonies were white and cottony, edge undulate, circular
growth appearance (upper side) and pale luteous (reverse side) (Fig. 1.C and 1.D). The acervuli
(average 280 μm) were black, concentric and appeared throughout the entire plate after 10 days.
Conidia were fusiform to ellipsoid and five-celled (average 22.7 × 7.7 μm) (n=100). Apical and
basal cells were hyaline, whereas the third and fourth cells from the base were darker (brown)
than the second cell from base (pale brown). Conidia had one basal appendage (average 9.5 μm)
and two to four apical appendages (average 25.9 μm) (Fig. 1.E). On the basis of colony and
conidia morphology, isolates were identified as Pestalotiopsis clavispora (G.F. Atk.) Steyaert,
recently renamed as Neopestalotiopsis clavispora (Maharachchikumbura et al. 2014). The
identification as P. clavispora was confirmed by amplifying and sequencing of three genetic
regions: ITS [(ITS1, 5.8S, ITS2) (Gen Bank accession nº KU096879), β-tubulin (KU096880) and tef1 gene (KU096881) from three isolates (Maharachchikumbura et al. 2012). BLAST
analysis showed in all cases 98 to 99% identity with P. clavispora [KM402033.1 (ITS),
KC537822.1 (β-tubulin), KM402034.1, JX399044 and KC537815.1 (tef1)]. Pathogenicity assays
were carried out in a randomized complete block design with three blocks of 12 plants each,
divided among three isolates and a control. Three isolates were compared to non-inoculated
controls on three strawberry cultivars: ‘Florida Radiance’, ‘Sabrina’ and ‘Splendor’. The roots of three strawberry transplants per isolate were dipped for 4 min in a conidial suspension (1·105
conidia·ml-1) from 10-day-old PDA cultures and planted in pots (14-cm diameter) filled with
sterilized soil. Control plants were dipped in sterilized 0.01% Tween-20. Plants were held at
25°C and 40/70% relative humidity (day/night) in a growth chamber with a 16-h photoperiod.
After 3 months of incubation, the mortality of the inoculated plants was: 7.4, 7.4 and 11.1% for
‘Florida Radiance’, ‘Splendor’ and ‘Sabrina’, respectively. No mortality was observed in the
control plants. About 23.4% and 25.9% of the inoculated plants showed more than 75% of root
rot and crown rot, respectively. The experiment was repeated twice. All isolates showed similar
pathogenicity. P. clavispora was consistently re-isolated from symptomatic plants. P. clavispora
has been reported causing canker and dieback on blueberry in Chile (Espinoza et al. 2008) and
Uruguay (González et al. 2012). P. longisetula has been reported as the cause of strawberry fruit
rot in Egypt (Embaby, 2007), but to our knowledge, this is the first report of P. clavispora
causing crown and root rot of strawberry worldwide.
A new species of Neopestalotiopsis based on both morphological and molecular characteristics is described. Neopestalotiopsis iranensis sp. nov. isolated from rotted strawberry (Fragaria
ananassa) fruits as well as from stolon and leaf lesions in Kurdistan province, Iran. Initially, light tan and sunken spots developed on fruits and resulted in a soft decay of the fruit flesh. The new species is morphologically distinguished from similar species with different conidium size and by possessing longer apical appendages, as well as some knobbed basal appendages. Phylogenetic analyses (Bayesian inference, maximum likelihood and maximum parsimony analyses) based on internal transcribed spacer, β-tubulin, and partial translation elongation factor 1-alpha combined gene sequences also indicated that this species is phylogenetically distinct from others. Moreover, strawberry crop is introduced here as a new host for N. mesopotamica.
We introduce molecularevolution.org, a publicly available gateway for high-throughput, maximum-likelihood phylogenetic analysis
powered by grid computing. The gateway features a garli 2.0 web service that enables a user to quickly and easily submit thousands of maximum likelihood tree searches or bootstrap
searches that are executed in parallel on distributed computing resources. The garli web service allows one to easily specify partitioned substitution models using a graphical interface, and it performs sophisticated
post-processing of phylogenetic results. Although the garli web service has been used by the research community for over three years, here we formally announce the availability of the
service, describe its capabilities, highlight new features and recent improvements, and provide details about how the grid
system efficiently delivers high-quality phylogenetic results. [garli, gateway, grid computing, maximum likelihood, molecular evolution portal, phylogenetics, web service.]
The genus Pestalotiopsis has received consider-able attention in recent years, not only because of its role as a plant pathogen but also as a commonly isolated endophyte which has been shown to produce a wide range of chemically novel diverse metabolites. Classification in the genus has been previously based on morphology, with conidial characters being considered as important in distinguishing species and closely related genera. In this review, Pestalotia, Pestalotiopsis and some related genera are evaluated; it is concluded that the large number of described species has resulted from introductions based on host association. We suspect that many of these are probably not good biological species. Recent molecular data have shown that conidial characters can be used to distinguish taxa; however, host association and geograph-ical location is less informative. The taxonomy of the genera complex remains confused. There are only a few type cultures and, therefore, it is impossible to use gene sequences in GenBank to clarify species names reliably. It has not even been established whether Pestalotia and Pestalotiopsis are distinct genera, as no isolates of the type species of Pestalotia have been sequenced, and they are morphologically somewhat similar. When selected GenBank ITS accessions of Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae are aligned, most species cluster throughout any phylogram generated. Since there appears to be no living type strain for any of these species, it is unwise to use GenBank sequences to represent any of these names. Type cultures and sequences are available for the recently described species P. hainanensis, P. jesteri, P. kunmingensis and P. pallidotheae. It is clear that the important species in Pestalotia and Pestalotiopsis need to be epitypified so that we can begin to understand the genus/genera. There are numerous reports in the literature that various species produce taxol, while others produce newly discovered compounds with medicinal potential and still others cause disease. The names assigned to these novel compound-producing taxa lack an accurate taxo-nomic basis, since the taxonomy of the genus is markedly confused. Until the important species have been epitypi-fied with living strains that have been sequenced and deposited in public databases, researchers should refrain from providing the exact name of species.
Pestalotiopsis is a taxonomically confused, pathogenic and chemically creative genus requiring a critical re-examination using a multi-gene phylogeny based on ex-type and ex-epitype cultures. In this study 40 isolates of Pestalotiopsis, comprised of 28 strains collected from living and dead plant material of various host plants from China were studied by means of morphology and analysis of ITS, β–tubulin and tef1 gene sequence data. Based on molecular and morphological data we describe 14 new species (Pestalotiopsis asiatica, P. chinensis, P. chrysea, P. clavata, P. diversiseta, P. ellipsospora, P. inflexa, P. intermedia, P. linearis, P. rosea, P. saprophyta, P. umberspora, P. unicolor and P. verruculosa) and three species are epitypified (P. adusta, P. clavispora and P. foedans). Of the 10 gene regions (ACT, β-tubulin, CAL, GPDH, GS, ITS, LSU, RPB 1, SSU and tef1) utilized to resolve cryptic Pestalotiopsis species, ITS, β–tubulin and tef1 proved to be the better markers. The other gene regions were less useful due to poor success in PCR amplification and/or in their ability to resolve species boundaries. As a single gene tef1 met the requirements for an ideal candidate and functions well for species delimitation due to its better species resolution and PCR success. Although β-tubulin showed fairly good differences among species, a combination of ITS, β-tubulin and tef1 gene data gave the best resolution as compared to single gene analysis. This work provides a backbone tree for 22 ex-type/epitypified species of Pestalotiopsis and can be used in future studies of the genus.
We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including
options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained
alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples
to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to
clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.
The genus Pestalotiopsis has received consider-able attention in recent years, not only because of its role as a plant pathogen but also as a commonly isolated endophyte which has been shown to produce a wide range of chemically novel diverse metabolites. Classification in the genus has been previously based on morphology, with conidial characters being considered as important in distinguishing species and closely related genera. In this review, Pestalotia, Pestalotiopsis and some related genera are evaluated; it is concluded that the large number of described species has resulted from introductions based on host association. We suspect that many of these are probably not good biological species. Recent molecular data have shown that conidial characters can be used to distinguish taxa; however, host association and geograph-ical location is less informative. The taxonomy of the genera complex remains confused. There are only a few type cultures and, therefore, it is impossible to use gene sequences in GenBank to clarify species names reliably. It has not even been established whether Pestalotia and Pestalotiopsis are distinct genera, as no isolates of the type species of Pestalotia have been sequenced, and they are morphologically somewhat similar. When selected GenBank ITS accessions of Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae are aligned, most species cluster throughout any phylogram generated. Since there appears to be no living type strain for any of these species, it is unwise to use GenBank sequences to represent any of these names. Type cultures and sequences are available for the recently described species P. hainanensis, P. jesteri, P. kunmingensis and P. pallidotheae. It is clear that the important species in Pestalotia and Pestalotiopsis need to be epitypified so that we can begin to understand the genus/genera. There are numerous reports in the literature that various species produce taxol, while others produce newly discovered compounds with medicinal potential and still others cause disease. The names assigned to these novel compound-producing taxa lack an accurate taxo-nomic basis, since the taxonomy of the genus is markedly confused. Until the important species have been epitypi-fied with living strains that have been sequenced and deposited in public databases, researchers should refrain from providing the exact name of species.
Since its introduction in 2001, MrBayes has grown in popularity as a software package for Bayesian phylogenetic inference using Markov chain Monte Carlo (MCMC) methods. With this note, we announce the release of version 3.2, a major upgrade to the latest official release presented in 2003. The new version provides convergence diagnostics and allows multiple analyses to be run in parallel with convergence progress monitored on the fly. The introduction of new proposals and automatic optimization of tuning parameters has improved convergence for many problems. The new version also sports significantly faster likelihood calculations through streaming single-instruction-multiple-data extensions (SSE) and support of the BEAGLE library, allowing likelihood calculations to be delegated to graphics processing units (GPUs) on compatible hardware. Speedup factors range from around 2 with SSE code to more than 50 with BEAGLE for codon problems. Checkpointing across all models allows long runs to be completed even when an analysis is prematurely terminated. New models include relaxed clocks, dating, model averaging across time-reversible substitution models, and support for hard, negative, and partial (backbone) tree constraints. Inference of species trees from gene trees is supported by full incorporation of the Bayesian estimation of species trees (BEST) algorithms. Marginal model likelihoods for Bayes factor tests can be estimated accurately across the entire model space using the stepping stone method. The new version provides more output options than previously, including samples of ancestral states, site rates, site d(N)/d(S) rations, branch rates, and node dates. A wide range of statistics on tree parameters can also be output for visualization in FigTree and compatible software.
There has been considerable disagreement regarding the relationships among Pestalotiopsis species and their delimitations. A molecular phylogenetic analysis was conducted on 32 species of Pestalotiopsis in order to evaluate the utility of morphological characters currently used in their taxonomy. Phylogenetic relationships were inferred from nucleotide sequences in the ITS regions and 5.8S gene of the rDNA under four optimality criteria: maximum parsimony, weighted parsimony, maximum likelihood, and neighbor joining. Phylogenies estimated from all analyses yielded trees of essentially similar topology and revealed 3 major groups that correspond with morphology-based classification systems. Molecular data indicated that the genus contains two distinct lineages based on pigmentation of median cells and four distinct groupings based on morphology of apical appendages. The analyses did not support reliability of other phenotypic characters of this genus, such as spore dimensions. Characters with particular phylogenetic significance are discussed in relation to the taxonomy of Pestalotiopsis.
Strawberry anthracnose, caused mainly by the Colletotrichum acutatum species complex, is a major disease in strawberry nurseries and production fields. The use of fungicides, such as the quinone outside inhibitors (QoIs), has been extensively deployed for the control of C. acutatum for the past 20 years. C. acutatum resistance to the QoIs was first reported in 2013 in Florida strawberry production fields. In 2015, anthracnose outbreaks were reported in strawberry nurseries and production fields across the United States. To elucidate the significance and geographical extension of C. acutatum resistance, fungicide use surveys were conducted, and isolates were collected in the affected areas. QoI-resistant isolates were collected from strawberry production fields and nurseries in six states in the United States. Fungicide use surveys indicated that, in some locations, the number of QoI applications exceeded the recommendation for this fungicide group. Thus, the current situation warrants immediate changes in anthracnose management strategies that integrate other chemical as well as nonchemical strategies to limit resistant population selection and prevent future anthracnose outbreaks.
A severe foliar disease was observed on leaves and fruits of strawberry (Fragaria × ananassa) for the first time from Indo-Gangetic plains of West Bengal in India during 2016–2017. Typical symptom appeared as a small necrotic lesion with a grey coloured centre and purplish brown margin. The spots were scattered on the leaves and gradually the spots coalesced to each other and covered large necrotic areas causing gradual conversion from green areas into black necrotic areas. As the spots were older and matured, black coloured acervuli developed from the necrotic portion. Morphologically the pathogen was very much similar with the genus Neopestalotiopsis previously reported from other countries. Through molecular characterization the pathogen in the present study was confirmed as Neopestalotiopsis clavispora (NCBI accession number MF377347). Further isolated pathogen was tested in some leading strawberry varieties of India. Although all the cultivars showed susceptibility under laboratory as well as field conditions, the variety “Sabrina” showed moderately resistance in leaves as well as fruits among the tested varieties.
収穫出荷されたブドウについて腐敗等の障害果実から糸状菌の分離を試みたところ,Botrytis cinerea, Cladosporium, Alternaria, Pestalotiopsis, Stemphyrium, Fusarium, Penicillium, Rhizopus, Aspergillus属菌が分離された。これらの糸状菌を健全なブドウの果粒に接種したところ,有傷接種では分離されたすべての糸状菌が,また,無傷接種でも,Cladosporium, Fusarium, Aspergillus属菌以外の分離菌は果粒の腐敗等の病徴を再現した。ブドウの障害果実からは2種類のPestalotiopsis属菌が分離され,それらは分生子の形態的特徴からP. menezesiana, P. uvicolaと同定された。また,両菌とも接種によってブドウの果実に腐敗症状を再現した。これらの2種のPestalotiopsis属菌は,ブドウ栽培圃場の未熟果実からも分離され,圃場に分布していることも確認された。圃場分離菌株を健全なブドウ果実に接種した場合にも,出荷ブドウ果実の腐敗と同様の症状が観察された。以上の結果から,ブドウ果実から分離されたP. menezesianaよびP. uvicolaは,ブドウ栽培圃場にも普遍的に分布しており,収穫出荷後においてブドウ果実に腐敗をもたらす病原菌となることが確認された。
A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing
methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform
(FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid
residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy
of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length.
Two different heuristics, the progressive method (FFT‐NS‐2) and the iterative refinement method (FFT‐NS‐i), are implemented
in MAFFT. The performances of FFT‐NS‐2 and FFT‐NS‐i were compared with other methods by computer simulations and benchmark
tests; the CPU time of FFT‐NS‐2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT‐NS‐i is over
100 times faster than T‐COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
Pestalotia leaf spot, caused by the fungus Pestalotiopsis longisetula Guba, has become the major disease affecting strawberry production in Brazil. Strawberry seedlings with 4–5 leaves were inoculated with a conidial suspension of P. longisetula (2 × 105 conidia/ml), and leaf samples were collected at 48, 72, 96 and 144 h after inoculation (hai) for observation in the scanning electron microscope. Conidia germinated within 48 hai. At 72 hai, conidia had formed very long germ tubes over the epidermal cells without any evidence of appressorial formation nor direct penetration. At 96 hai, fungal hyphae grew inter- and intracellularly in the lacunous parenchyma and also through tracheary elements. Pycnidia were first observed on the leaf surface at 96 hai. At 144 hai, conidia of P. longisetula were first liberated from the pycnidia. This study adds new information to better understand of the infection process of P. longisetula that may help in developing more effective disease control strategies.
Cold-stored plants of strawberry cultivars Tamella, Cambridge Favourite and Redgauntlet were more susceptible to pathogenic isolates of Phytophthora cactorum than similar plants which had not been cold-stored. Indigenous nonpathogenic isolates of P. cactorum did not cause crown rot in cold-stored plants, although a small number of symptomless latent infections occurred. The majority of P. cactorum isolates causing crown rot symptoms were taken from infected strawberry crowns, although two isolates from gooseberry plants, but of uncertain origin, were also pathogenic. Outbreaks of crown rot in areas with no previous history of the disease therefore probably result from the importation of non-indigenous inoculum with planting material.
Assessments of the timing of infection in relation to cold storage revealed that a high incidence of death in the cold store and chronic wilt symptoms on planting from the store resulted from initiating symptomless infections prior to cold storage. However, infection during the period immediately after cold storage resulted in rapid wilt symptoms of Phytophthora crown rot. When plated in sterile distilled water for 24 h, pieces of tissue from infected plants which had died during cold storage produced large numbers of sporangia and zoospores. This indicates that such plant material could provide a potent source of inoculum for infections in the post storage thawing environment. It is proposed that a combination of heightened host susceptibility resulting from cold storage and the presence of scatted latent infections or infected debris among the plants could result in a sudden, large scale appearance of crown rot, as sometimes is seen with cold-stored plantings of strawberries.
Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/31361/1/0000273.pdf
We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.
A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
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