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Primers and probes used for the quantitative real-time PCR (qPCR) detection of Pythium ultimum var. ultimum, P. irregulare, P. vexans and the genus Phytophthora
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Replant and decline diseases of grapevines not only cause quantitative and qualitative yield losses, but also result in extra
costs when vineyards have to be replanted. This study investigated the role of Pythium and Phytophthora in the decline syndrome in South Africa by determining (1) the species associated with nursery and established vines, an...
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Citations
... As the target root pathogens G. irregulare and F. pseudograminearum are known to actively colonize wheat roots throughout the growing season (Cook 2001, Paulitz et al. 2002, inoculant rhizosphere competence and persistence are key considerations when selecting strains for field suppression of Pythium root rot and Fusarium crown rot (Martin andLoper 1999 , Kazan andGardiner 2018 ). Species-specific quantitative polymerase chain reaction (qPCR) assays for G. irregulare (Spies et al. 2011 ), F. pseudograminearum (Obanor and Chakraborty 2014 ), T. gamsii , and T. harzianum (Stum-mer et al. 2020 ) have been used to monitor abundance and persistence of the pathogens and inoculant strains in soil and plant tissues. In controlled environments, qPCR indicated that strain A5MH exhibited greater wheat rhizosphere competence and in planta suppression of F. pseudograminearum than T. gamsii strain Tk7a and T. harzianum strains Tr904 and Tr906 (Stummer et al. 2020, Stummer et al. 2022 and was therefore selected for crown rot suppressive field trials. ...
... Tric hoderma g amsii A5MH, F. pseudograminearum , and G. irregulare DNA were quantified with qPCR assays Tk7a-A106, Fptri3e, and Pir, respectively (Spies et al. 2011, Obanor and Chakraborty 2014, Stummer et al. 2020. All qPCR assays were conducted on a CFX96 Real-Time PCR Detection System (Bio-Rad, C A, US A). ...
... Each 20 μl reaction consisted of 2-5 μl of DNA template (1 fg-10 ng) and 10 μl of 2 × SsoFast™ EvaGreen ® Supermix (Bio-Rad, C A, US A). Assays Tk7a-A106 and Fptri3e used 20 pMol of each primer per reaction, whereas assay Pir used 6 pMol of primer PirF1 and 18 pMol of primer PirR3 (Spies et al. 2011, Moein et al. 2019 ). The qPCR cycling conditions were 95 • C for 3 min, followed by 40 cycles of denaturation at 95 • C for 10 s and annealing for 30 s at 60 • C (Tk7a-A106), 59 • C (Fptri3e), or 65 • C (Pir). ...
Aims
Determine the wheat rhizosphere competence of Trichoderma gamsii strain A5MH and in planta suppression of the Pythium root and Fusarium crown rot pathogens Globisporangium irregulare and Fusarium pseudograminearum.
Methods and results
Wheat was continuously cropped (eight years) at a minimum tillage, low growing season rainfall (GSR ≤ 170 mm) site shown as highly conducive to Pythium root and Fusarium crown rots. Root isolation frequency (RIF) and qPCR were used to determine the rhizosphere dynamics of strain A5MH and the target pathogens at tillering, grain harvest, and in postharvest stubble over the final 2 years. Strain A5MH actively colonized the wheat rhizosphere throughout both growing seasons, had high root abundance at harvest [log 4.5 genome copies (GC) g−1] and persisted in standing stubble for at least 293-d postinoculation. Globisporangium irregulare was most abundant in roots at tillering, whereas F. pseudograminearum was only abundant at harvest and up to 9-fold greater in the drier, second year (GSR 105 mm). Strain A5MH decreased RIF of both pathogens by up to 40%, root abundance of G. irregulare by 100-fold, and F. pseudogaminearum by 700-fold, but was ineffective against crown rot in the second year when pathogen abundance was >log 6.0 GC g−1 root. Strain A5MH increased crop emergence and tillering biomass by up to 40%.
Conclusions
Further trials are required to determine if the A5MH-induced pathogen suppression translates to yield improvements in higher rainfall regions where non-cereal rotations reduce crown rot inoculum.
... These characteristics altogether indicate that the genus Phytopythium presents morphological and phylogenetic criteria that are intermediate between Pythium and Phytophthora (Bala et al. 2010). Phytopythium was reported to be an important genus in agricultural production and several species are soil-and water-borne plant pathogens causing root rot and stem rot diseases on important plants like grapevine, macadamia, kiwi, holm oak, buckwheat, avocado, apple, pear and Shatangju mandarin (Belbahri et al. 2008;Spies et al. 2011;Baten et al. 2014;Chen et al. 2016;Polat et al. 2017;Rodriguez-Padron et al. 2018Donati et al. 2020;Jabiri et al. 2020Jabiri et al. , 2021Tkaczyk 2020). A recent investigation conducted in South Africa at five apple nurseries over two years revealed the presence of Globisporangium irregulare and G. ultimum. ...
Considerable losses of apple and peach tree seedlings were observed on 2012 in Tunisian nurseries and orchards. In the current study, surveys were conducted on apple and peach seedlings in ten and six nurseries respectively. Sampling was undertaken from symptomatic roots of seedlings showing various levels of decline. Isolation studies revealed the presence of Globisporangium ultimum (10.46% of apple seedlings and 18.96% of peach seedling), Phytopythium mercuriale (7.79% of apple and 22.29% of peach seedlings), and Phytopythium helicoides (5.8% of apple seedlings). On apple, a detached twig assay on the ‘Golden Delicious’ variety showed that P. mecuriale caused smaller necrotic lesions than G. ultimum and P. helicoides. A stem inoculation assay on the same variety supported the high virulence of G. ultimum which caused the death of seedlings. On the apple rootstock ‘MM106ʼ a soil inoculation assay supported the stem inoculation assay in that G. ultimum was the most virulent causing root rot and root weight reductions, followed by P. helicoides and P. mercuriale having the lowest virulence. On peach, a detached twigs essay on the ‘Carnival’ variety showed that G. ultimum and P. mercuriale caused necrotic lesions. A stem inoculation assay on both ‘Carnival’ and ‘Royal Glory’ varieties supported the virulence of these two oomycetes species which caused stem necrosis. On the peach rootstock ‘Garnem’ a soil inoculation assay showed that only G. ultimum was virulent causing root rot. These results suggest that these pathogens could function as potential decline inoculum sources that might limit post-plant tree growth in orchards.
... aff. monospermum has been isolated from grapevine [73], and S. anisospore is generally known as an aquatic pathogen [74]. The pathogenicity of these species in wheat soils is currently unknown. ...
Soil-borne oomycetes include devastating plant pathogens that cause substantial losses in the agricultural sector. To better manage this important group of pathogens, it is critical to understand how they respond to common agricultural practices, such as tillage and crop rotation. Here, a long-term field experiment was established using a split-plot design with tillage as the main plot factor (conventional tillage (CT) vs. no till (NT), two levels) and rotation as the subplot factor (monocultures of soybean, corn, or wheat, and corn–soybean–wheat rotation, four levels). Post-harvest soil oomycete communities were characterized over three consecutive years (2016–2018) by metabarcoding the Internal Transcribed Spacer 1 (ITS1) region. The community contained 292 amplicon sequence variants (ASVs) and was dominated by Globisporangium spp. (85.1% in abundance, 203 ASV) and Pythium spp. (10.4%, 51 ASV). NT decreased diversity and community compositional structure heterogeneity, while crop rotation only affected the community structure under CT. The interaction effects of tillage and rotation on most oomycetes species accentuated the complexity of managing these pathogens. Soil and crop health represented by soybean seedling vitality was lowest in soils under CT cultivating soybean or corn, while the grain yield of the three crops responded differently to tillage and crop rotation regimes.
... aff. monospermum has been isolated from grapevine [65], and S. anisospore is generally reported as an aquatic pathogen [66], their pathogenicity are unknown. We did not observe a significant effect of rotation on the beta-diversity of the soil oomycete community under NT. ...
Soil-borne oomycetes include devastating plant pathogens that cause substantial losses in the agricultural sector. To better manage this important group of pathogens, it is critical to understand how they respond to common agricultural practices, such as tillage and crop rotation. Here, a long-term field experiment was established using a split-plot design with tillage as the main plot factor (conventional tillage [CT] vs. no till [NT], 2 levels) and rotation as the subplot factor (monocultures of soybean, corn, or wheat, and corn-soybean-wheat rotation, 4 levels). Post-harvest soil oomycete communities were characterized over three consecutive years (2016-2018) by metabarcoding the Internal Transcribed Spacer 1 (ITS1) region. The community contained 292 Amplicon Sequence Variants (ASVs) and was dominated by Globisporangium spp. (85.1% in abundance, 203 ASV) and Pythium spp. (10.4%, 51 ASV). NT decreased diversity and community compositional structure heterogeneity, while crop rotation only affected the community structure under CT. The interaction effects of tillage and rotation on most oomycetes species accentuated the complexity of managing these pathogens. Soil and crop health represented by soybean seedling vitality was lowest in soils under CT cultivating soybean or corn, while grain yield of the three crops responded differently to tillage and crop rotation regimes.
... The presence of multiple divergent copies of the ITS region is a well-known character among Pythium s.l. species, such as Phytopythium helicoides, Pp. litorale, Pp. mercuriale, Pp. vexans complex, G. mamillatum, G. splendens, G. heterothallicum, G. rostratifingenes, G. irregulare, G. cryptoirregulare, G. ultimum, G. spinosum, G. perplexum, P. acanthicum complex, P. arrhenomanes and P. graminicola (Kageyama et al. 2007, Belbahri et al. 2008, McLeod et al. 2009, Spies et al. 2011). ...
During a survey of gardens in Shiraz County, Iran, aimed at identifying oomycetes associated with roots of ornamental trees, a species of Globisporangium with distinctive morphological characters separating it from other known species in this genus was recovered from conifers and occasionally from a Quercus sp. Five isolates of this species were characterised. Phylogenetic analyses of nuclear (ITS and βtub) and mitochondrial (cox1 and cox2) loci using Bayesian inference and maximum likelihood analyses as well as their distinct morphological and cultural characteristics (e.g., abundant production of chlamydospores; globose, ellipsoid to ovoid sporangia; amorphous oogonia with a smooth wall; paragynous to rarely hypogynous antheridia and 1–5 antheridia per oogonium; mostly plerotic oospores) revealed that these isolates belong to a new Globisporangium species grouping in the phylogenetic clade G of Pythium sensu lato. This paper formally describes Globisporangium coniferarum sp. nov. as a new species and compares it with other phylogenetically related and already known Globisporangium species, including G. nagaii, G. violae, G. paddicum, G. okanoganense, G. iwayamae and G. canariense.
Citation: Salmaninezhad F, Aloi F, Pane A, Mostowfizadeh-Ghalamfarsa R, Cacciola SO (2022). Globisporangium coniferarum sp. nov., associated with conifers and Quercus spp. Fungal Systematics and Evolution 10: 127–137. doi: 10.3114/fuse.2022.10.05
... The presence of multiple divergent copies of the ITS region is a well-known character among Pythium s.l. species, such as Phytopythium helicoides, Pp. litorale, Pp. mercuriale, Pp. vexans complex, G. mamillatum, G. splendens, G. heterothallicum, G. rostratifingenes, G. irregulare, G. cryptoirregulare, G. ultimum, G. spinosum, G. perplexum, P. acanthicum complex, P. arrhenomanes and P. graminicola (Kageyama et al. 2007, Belbahri et al. 2008, McLeod et al. 2009, Spies et al. 2011). ...
During a survey of gardens in Shiraz County, Iran, aimed at identifying oomycetes associated with roots of ornamental trees, a species of Globisporangium with distinctive morphological characters separating it from other known species in this genus was recovered from conifers and occasionally from a Quercus sp. Five isolates of this species were characterised. Phylogenetic analyses of nuclear (ITS and βtub) and mitochondrial (cox1 and cox2) loci using Bayesian inference and maximum likelihood analyses as well as their distinct morphological and cultural characteristics (e.g., abundant production of chlamydospores; globose, ellipsoid to ovoid sporangia; amorphous oogonia with a smooth wall; paragynous to rarely hypogynous antheridia and 1–5 antheridia per oogonium; mostly plerotic oospores) revealed that these isolates belong to a new Globisporangium species grouping in the phylogenetic clade G of Pythium sensu lato. This paper formally describes Globisporangium coniferarum sp. nov. as a new species and compares it with other phylogenetically related and already known Globisporangium species, including G. nagaii, G. violae, G. paddicum, G. okanoganense, G. iwayamae and G. canariense.
... Our study evaluated only pathogen presence, not density; additional studies, over longer time periods using molecular methods (e.g. Spies et al., 2011) would enable examination of pathogen density over time since tree loss. ...
Soil-borne pathogens can shape forest communities by lowering seedling survivorship. Many soil pathogens can persist long-term as survival spores, but how long pathogens outlive tree hosts in gap soils and whether they continue to affect seedling survival is uncertain. We studied the presence of oomycetes and evaluated seedling performance in soils near live Prunus serotina trees, and 0.5 and 1.5-y-old stumps. We isolated five species of oomycetes from soils, two of which were pathogenic (Pythium intermedium and Pythium irregulare) to Prunus serotina. There was a non-significant ~10.5% increase in conspecific seedling survival in stumps versus live trees, and pathogens were present in soils of all stump ages. The continued presence of pathogens of Prunus serotina in gap soils demonstrates the potential for impacts on conspecific regeneration after tree death, though the slight improvement in survival suggests that these effects may weaken with time.
... According to sequence accession data, it has also been isolated from the woody ornamentals Metrosideros and Schefflera in southern California nursery settings (HQ261700 and HQ261699). Although this pathogen can affect a broad range of ornamentals, and native plants in their natural ecosystems, it is also a pathogen and potential threat to agricultural woody plants, affecting grapevines (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in California and Spain (Browne et al. 2015;P erez-Sierra et al. 2010;Spies et al. 2011). In almond, P. niederhauserii causes crown rot, trunk, and scaffold cankers and ultimately wilting, defoliation, and tree death (Browne et al. 2015;P erez-Sierra et al. 2010). ...
... nursery potted ornamental plants in Europe and Australia, grapevine nursery material in South Africa, and severe decline of young almond trees in field-grown nurseries in Spain (Cacciola et al. 2008;Davison et al. 2006;P erez-Sierra et al. 2010;Prigigallo et al. 2015;Spies et al. 2011). The association of Phytophthora species with pistachio planting material in California has not been determined. ...
Pistachio is one of the most widely cultivated nut crops in California with approximately 115,000 hectares of bearing pistachio trees. In recent years, several orchards were identified with declining trees leading to substantial tree losses. Symptoms included trees with poor vigor, yellowing and wilting of leaves, crown rot and profuse gumming on the lower portion of trunks. Thirty-seven Phytophthora-like isolates were obtained from crown rot tissues in the rootstock of grafted pistachio trees and characterized by means of multi-locus phylogeny comprising ITS rDNA, beta-tubulin and mt cox1 sequence data. The analysis provided strong support for the delineation and identification of three Phytophthora species associated with declining pistachio trees, including Phytophthora niederhauserii, P. mediterranea and P. taxon walnut. Pathogenicity studies in potted UCBI rootstocks (clonal and seeded) confirmed that all three Phytophthora species can cause crown and root rot of pistachio, thus fulfilling Koch’s postulates. The widespread occurrence of Phytophthora crown rot in recently planted pistachio orchards and the high susceptibility of UCBI rootstocks suggest this disease constitute an emerging new threat to the pistachio industry of California. To the best of our knowledge, this study is the first to report P. niederhauserii, P. mediterranea and P. taxon walnut as causal agents of crown and root rots of pistachio.
... A new genus of Phytopythium, a relative of Phytophthora and Pythium has been reported [14] as a pathogen of several fruit trees, including citrus in Tunisia [15], kiwifruit in Turkey [16], avocados in the Canary Islands [17,18], and grapevines in South Africa [19], and isolated from freshwater environments in Korea [20]. Similar symptoms to those described previously were seen in apple and pear trees in Morocco. ...
... The pathogenic oomycete Pp. vexans was reported on apple trees in South Africa in 2011 [5] and on citrus in China [38] and Tunisia in 2017 [15]. It was also isolated from infected avocado trees in the Canary Islands [17], on avocado in Mexico [39], on kiwi in Turkey [16], on grapevines in South Africa [19], on cassava in Brazil [40], and in Vietnam on durian [41]. Furthermore, the analysis of the phylogenetic tree underlined a strong similarity of our isolates of Pp. vexans with those of different countries mentioned above. ...
An extensive survey conducted in the Saïss plain of Morocco during the 2017-2018 growing season revealed that 35 out of 50 apple and pear orchards were infested with a pathogen that causes the decline disease. Morphological and phylogenetic tree analyses using the cox II gene allowed us to identify the pathogen as Phytopythium vexans. Interestingly, no Phytophthora and Pythium species were isolated. The occurrence and prevalence of the disease varied between locations ; the most infested locations were Meknes (100%), Imouzzer (83%), and Sefrou (80%). To fulfill Koch's postulate, a greenhouse pathogenicity test was performed on the stem and collar of one-year-old healthy seedlings of apple rootstock M115. Symptoms similar to those observed in the field were reproduced in less than 4 months post-inoculation with root rot disease severity ranging from 70 to 100%. The survey results evidenced that apple rootstocks, soil type, and irrigation procedure may contribute significantly to the occurrence of the disease. The disease was most prevalent in drip water irrigation and sandy-clay soil on wild apple rootstock. Accordingly, a rational drip advanced watering system and good sanitation practices could eliminate water stagnation and help prevent the onset of this disease. It was concluded that Pp. vexans occurrence may be strongly influenced by irrigation mode and type of soil. Therefore, the obtained findings of this study could help to better understand the recurrence of this disease and to develop a reliable integrated strategy for its management .
... Mycelia were harvested, collected, and stored at −20 • C. Mycelial DNA was extracted by a Rapid Extraction Kit for Fungi Genomic DNA (Aidlab, Beijing, China) following the user's manual. Conventional PCR amplification was carried out with the specific primers of P. vexans (PvF1/PvR1) (Spies et al., 2011b). The ITS region (ITS1, 5.8S, and ITS2) of all 20 isolates was amplified and sequenced using primers V9G (5 -TTACGTCCCTGCCCTTTGTA-3 ) and LS266 (5 -GCATTCCCAAACAACTCGACTC-3 ) (van den Ende and de Hoog, 1999). ...
Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.
