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Peronospora tabacina is an obligate parasite that causes blue mold of tobacco. The pathogen reproduces primarily by sporangia, whereas the sexual oospores are rarely observed. A collection of 122 isolates of P. tabacina was genotyped using nine microsatellites to assess the population structure of individuals from subpopulations collected from central, southern, and western Europe; the Middle East; Central America; North America; and Australia. Genetic variations among the six subpopulations accounted for ∼8% of the total variation, including moderate levels of genetic differentiation, high gene flow among these subpopulations, and a positive correlation between geographic and genetic distance (r = 0.225; P < 0.001). Evidence of linkage disequilibrium (P < 0.001) showed that populations contained partially clonal subpopulations but that subpopulations from Australia and Mediterranean Europe did not. High genetic variation and population structure among samples could be explained by continuous gene flow across continents via infected transplant exchange and/or long-distance dispersal of sporangia via wind currents. This study analyzed the most numerous P. tabacina collection and allowed conclusions regarding the migration, mutation, and evolutionary history of this obligate biotrophic oomycete. The evidence pointed to the species origin in Australia and identified intracontinental and intercontinental migration patterns of this important pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

An outbreak of downy mildew disease of onion, caused by Peronospora destructor, in Japan in 2016 necessitated a reevaluation of the primary inoculum sources to optimize disease management. Detection of the P. destructor pathogen in plants with asymptomatic infection and in soil would guide the application of fungicides according to the extent of infection before disease development. Here, we detected P. destructor in both plants and soil using newly developed primer sets (Pd ITS and Pd ITS 614) by both conventional and real-time PCR. Validation by real-time PCR with Pd ITS 614 showed that P. destructor DNA was amplified from symptomless seedlings at 3.7 × 102 to 1.0 × 100 conidium cells/50 mg leaf tissue, suggesting the detection of asymptomatic infection. Real-time PCR with Pd ITS amplified pathogen DNA from field soils at 1.6 × 103 to 8.3 × 101 oospore cells/g of soil. This real-time PCR assay provides a useful tool for identifying and quantifying inoculum sources, which may be the foundation of the design of integrated disease management strategies.

Late the blight of potato is a devastating and one of the economic diseases of potato and other plants belonging to family Solanaceae. Late blight, caused by Phytophthora infestans, is one of the most threatening pathogenic diseases which not only results in direct crop losses but also cause farmers to embrace huge monetary expenses for disease control and preventive measure. It was first reported during the Irish Potato Famine, leading to massive starvation in Ireland and other parts of Europe during the middle of 19th century. Phytopthora harms the foliar portion in the field and also the tuber in the storage that can result in complete crop failure in potato. The pathogen has distinct survival mechanisms and two life cycles infection processes. The development of a sexual spore known as oospore includes two types of pairs, A1 and A2. The spores are introduced to good plants by wind and rain. Different methods for prevention of crops from late blight has been developed and used worldwide. An integrated disease management strategy includes successful control of this disease. Cultural control, chemical management, and advanced disease management are the most effective interventions. Integration of late blight control in tropical regions with abundant fungal inoculants in most months of the year was also seen as one of the best choices in disease management. This paper reviews the significance of late blight of potato and controlling strategies adopted for minimizing yield losses incurred by this disease by the application of synthetic fungicides and different organic amendments.

Onion downy mildew (ODM), caused by Peronospora destructor, is a serious threat for onion growers worldwide. In southwestern Québec, Canada, a steady increase in occurrence of ODM has been observed since the mid-2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which act as initial inoculum locally and also for neighboring areas. It also produces oospores capable of surviving in soils and tissues for a prolonged period of time. A recent study showed that ODM epidemics are strongly associated with weather conditions related to production and survival of overwintering inoculum, stressing the need to understand the role of primary (initial) and secondary inoculum. However, P. destructor is an obligate biotrophic pathogen, which complicates the study of inoculum sources. This study aimed at developing a molecular assay specific to P. destructor, allowing its quantification in environmental samples. In this study, a reliable and sensitive hydrolysis probe-based assay multiplexed with an internal control was developed on the internal transcribed spacer (ITS) region to quantify soil- and airborne inoculum of P. destructor. The assay specificity was tested against 17 isolates of P. destructor obtained from different locations worldwide, other members of the order Peronosporales, and various onion pathogens. Validation with artificially inoculated soil and air samples suggested a sensitivity of less than 10 sporangia g−1 of dry soil and 1 sporangium m−3 of air. Validation with environmental air samples shows a linear relationship between microscopic and real-time quantitative PCR counts. In naturally infested soils, inoculum ranged from 0 to 162 sporangia equivalent g−1 of dry soil, which supported the hypothesis of overwintering under northern climates. This assay will be useful for primary and secondary inoculum monitoring to help characterize ODM epidemiology and could be used for daily tactical and short-term strategic decision-making.

The enigmatic microfossil Triskelia scotlandica from the Lower Devonian Rhynie chert is an acritarch-like, usually spheroidal to ovoid vesicle with a surface ornamentation comprised of isolated triradiate and meandering, high membranous ridges. The form has been formally described based on dispersed specimens preserved inside the remains of a land plant axis, and interpreted as the resting stage of a microscopic alga. Unusual microbial coatings on land plant axes from the Rhynie chert have recently yielded > 300 additional specimens of T. scotlandica, many of which occur in situ in prominent swellings of fungal hyphae. This discovery is a strong evidence that T. scotlandica is not algal, but rather fungal or fungus-like in nature. The swellings might be oogonia of a representative of the Oomycota (e.g., Saprolegniales), and T. scotlandica accordingly an ornamented oospore. However, specimens with a discharge tube suggest that T. scotlandica is more likely a zoosporangium or resting spore stage of an endoparasite, perhaps with affinities to the holocarpic Oomycota (e.g., Olpidiopsis), Cryptomycota (e.g., Rozella), or zoosporic fungi (e.g., Olpidium), in which case the hyphal swellings would be either dilatations resulting from the expansion of the parasite inside, or a host response (hypertrophy). The affinity of T. scotlandica remains unresolved, due in part to the uncertain diagnostic value of the surface ornamentation, and because no other stages of the life history of this organism are known. Nevertheless, the new specimens expand our knowledge of the microbial interactions that existed in early terrestrial ecosystems.

Phytophthora root rot (PRR) caused by Phytophthora medicaginis (Pm) is an important disease of chickpea in Australia. There are limited control options, with avoiding planting chickpeas in paddocks with a high PRR risk a key management strategy. Currently, risk assessment is based solely on paddock history of PRR, without any measure of Pm inoculum. We developed a qPCR test to quantify Pm inoculum concentrations in soil and evaluated its ability to predict, prior to planting, PRR disease and yield loss in chickpea. The qPCR test was specific to Pm and did not cross react with other Phytophthora species found in Australian agricultural systems and was sensitive, being able to detect

Downy mildew of spinach, caused by the obligate pathogen Peronospora effusa, remains the most important constraint in the major spinach production areas in the United States. This disease can potentially be initiated by asexual sporangiospores via “green bridges”, sexually derived oospores from seed or soil, or dormant mycelium. However, the relative importance of the various types of primary inoculum is not well known. The ability of P. effusa sporangiospores to withstand abiotic stress, such as desiccation, and remain viable during short- and long-distance dispersal and the ability of oospores to germinate and infect seedlings remain unclear. Thus, the primary objectives of this research were to evaluate the impact of desiccation on sporangiospore survival and infection efficiency and examine occurrence, production, and germination of oospores. Results indicate that desiccation significantly reduces sporangiospore viability as well as infection potential. Leaf wetness duration of 4 h was needed for disease establishment by spinach downy mildew sporangiospores. Oospores were observed in leaves of numerous commercial spinach cultivars grown in California in 2018 and Arizona in 2019. Frequency of occurrence varied between the two states-years. The presence of opposite mating types in spinach production areas in the United States was demonstrated by pairing isolates in controlled crosses and producing oospores on detached leaves as well as intact plants. Information from the study of variables that affect sporangiospore viability and oospore production will help in improving our understanding of the epidemiology of this important pathogen, which has implications for management of spinach downy mildew.

A stem rot disease appeared on hydrangea plants in Tochigi Prefecture, Japan. Four Phytophthora-like isolates were obtained, and a representative isolate was selected for analysis. Hydrangea plants inoculated with the isolate developed similar symptoms. Molecular phylogenetic analyses using the rDNA ITS region and cox1, EF1-α, and β-tubulin genes indicated that the isolate was Phytophthora hedraiandra. The sporangia were papillate, laterally sessile, and caducous. Antheridia were paragynous or amphygynous, and laterally sessile, and the oospores were aplerotic. Based on these characteristics, the isolate was identified as P. hedraiandra. We suggest naming this hydrangea disease “Ajisai kukieki byo”.

Made available in DSpace on 2022-05-01T09:30:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 During the winter, there is a high occurrence of downy mildew on lettuce caused by Bremia lactucae. This oomycete shows variability in virulence, so understanding the genetic structure of the pathogen population becomes essential for obtaining resistant cultivars. Thus, the objective of this study was to determine sexual compatibility in Brazilian populations of B. lactucae and investigate the occurrence of sexual reproduction of the pathogen on lettuce (Lactuca sativa) and prickly lettuce (Lactuca serriola). Leaf samples were collected in 33 municipalities in seven Brazilian states. The virulence structure of the populations was monitored using the EU-C sextet code. B. lactucae populations from the states of São Paulo, Paraná, Rio de Janeiro, and Rio Grande do Sul shared six of the 15 virulence factors evaluated. Twenty-five virulence phenotypes (v-phenotypes) were found, with the sextet codes 31-00-02, 31-16-02, 31-24-02, and 31-01-02 being more frequent. The predominance of some v- phenotypes indicates that clonal reproduction is still the main form of B. lactucae propagation. The genes and resistance factors of the cultivars Argelès (Dm38), Balesta, and Bartoli are recommended as suitable sources of lettuce resistance in Brazil. Natural occurrence of oospores was detected in most sampled locations, in lettuce and prickly lettuce plants. Virulence variability of Brazilian isolates is the result of the pathogen's ability to reproduce both sexually and asexually, with a prevalence of homothallic isolates; although the majority were of the predominant B2 mating type, there was a high incidence of predominant B1 in addition to B1 = B2. Department of Agricultural Production Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences, SP Department of Plant Pathology Gulf Coast Research and Education Center University of Florida Research Department – Lettuce Breeding Agristar do Brasil, SP Department of Botany Faculty of Science Palacký University in Olomouc Department of Agricultural Production Sciences São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences, SP

International audience; Aphanomyces euteiches Drechs is a pathogenic soil-borne oomycete that causes root rot of legumes, one of the most serious diseases affecting legume production worldwide. There is currently no registered pesticide and no available resistant pea cultivar. Avoidance of infested fields based on disease risk assessment prior to pea sowing remains the main method available to manage the disease, but time-consuming bioassays are required to assess disease severity on susceptible plants grown in field soil samples. Direct quantification of A. euteiches inoculum in the soil by targeting multicopy genomic sequences of the internal transcribed spacer 1 with a qPCR-based method has been proposed as a rapid alternative for disease prediction. However, the method lacks sensitivity to accurately quantify low inoculum levels from naturally infested fields. We developed a suitable methodology based on droplet digital PCR (ddPCR) to quantify low A. euteiches inoculum levels in naturally infested soils. The methodology was validated on 200 soil samples taken from four naturally infested fields in the main pea cropping area in the north of France. The comparative analysis of inoculum density and disease severity of the 50 samples within each of the four fields revealed a non-homogeneous distribution of the A. euteiches population; this explains why the disease is visible in the form of foci. A significant relationship between pea root rot disease severity determined by bioassays and A. euteiches inoculum density was highlighted, and a linear mixed model is proposed to predict disease severity from inoculum density.

Phytophthoraare eukaryotic microbes that cause disease in a wide range of agriculturally and ecologically important plants. During thePhytophthoradisease cycle, thick-walled oospores can be produced via sexual reproduction. These resting spores can survive in the soil for several years in the absence of a host plant, thus providing a long-term inoculum for disease. The ability to quantitatively evaluate oospore viability is an important part of many phytopathology studies. Here, we tested six fluorescent viability dyes for their ability to differentially stainPhytophthora agathidicidaoospores: SYTO 9, FUN-1, fluorescein diacetate (FDA), 5-carboxyfluorescein diacetate (CFDA), propidium iodide, and TOTO-3 iodide. Each dye was first tested individually with untreated or heat-treated oospores as proxies for viable and non-viable oospores, respectively. SYTO9, FUN-1, CFDA and propidium iodide stained untreated and heat-treated oospores indiscriminately. In contrast, FDA (a green-fluorescent viable cell stain) and TOTO-3 (a red-fluorescent non-viable cell stain) differentially stained untreated or heat-treated oospores with no cross-fluorescence. We then tested the efficacy of dual viability staining and in conjunction with a pipeline for automated image analysis. To validate the method, untreated and heat-treated oospores were mixed at specific ratios, dual-stained, and analyzed using the pipeline. Linear regression of the resulting data showed a clear correlation between the expected and measured oospore ratios (dy/dx=0.95,R2=0.88). Overall, the combination of dual-fluorescence staining and automated image analysis provides a high-throughput method for quantitatively assessing oospore viability and therefore can facilitate further studies on this key part of thePhytophthoradisease cycle.

Alder dieback remains a major problem in European alder stands and its spread continues to threaten their existence. The causal agent of this disease is the so-called alder Phytophthora species complex, which includes the hybrid Phytophthora ×alni and its parental species P. uniformis and P. ×multiformis. Little is known about the survival of these Phytophthora species in alder. The aim of our investigations was to find out whether, and if so where, the pathogen survives. The subject of these studies was alder roots. Therefore, artificial infection studies and histological studies with P. ×alni and P. uniformis were carried out on seedlings of black alder (Alnus glutinosa). These histological studies revealed oogonia and oospores of P. ×alni and P. uniformis in different parts of the root tissue.

is an important root pathogen of several legume and forage crops worldwide. Disease management is challenging due to resilient oospores that can persist in the soil for long periods of time. Several research groups are working towards incorporating disease resistance into crop cultivars involving large-scale disease resistance screening and, for some crops, the use of molecular markers associated with quantitative trait loci for resistance. The refinement of laboratory protocols for efficient pathogen isolation, reliable and simple zoospore production, and a repeatable indoor screening assay are important tools needed to further research in this area. The standardization of these protocols among research groups would provide an efficient platform for research collaboration. The effect of different media and sporulation induction methods on zoospore production was assessed. The most effective and repeatable method of A. euteiches zoospore production involved culturing and incubation for 4 days on autoclaved wheat leaf segments placed on corn meal-yeast extract-phosphate buffer agar. Colonized wheat leaves were transferred into 100 mL of distilled water in 250 mL flasks and incubated at 100 rpm for 18 h at 24ºC to induce zoospore production. Five zoospore concentrations ranging from 100 to 10 000 zoospores mL−1 were evaluated using susceptible and partially resistant pea and lentil genotypes to optimize zoospore concentration for germplasm screening. The best differentiation of levels of resistance among lentil and pea genotypes was achieved with 1000 zoospores mL−1.

Ginger (ZingiberofficinaleL.) belongs to family Zingiberaceae is an important oriental spice crop. Ginger is high value crop which grows well in warm and humid climate and is cultivated from sea level to an altitude of 1500 meters above sea level (Kandinnan, et al. 1996). It has special significance for tropical countries where it is produced and consumed in large quantities (Islam, 2017; BARI, 2012, 2013 and 2014). The aromatic rhizomes are used as spice and medicine. Major producers of ginger in the world are India, Jamaica, Sierra Leone, Nigeria, China, Japan, Taiwan and Australia (Rana and Sharma, 1998). Ginger is much more used in Bangladesh as a spice and is cultivated more or less all over the country. In the country produced only 74380 metric tons of ginger from 9120 ha of land and the yield per hectare was 8.15 kg (BBS, 2011). But average yield is low as compared to other ginger growing countries of the world. The production is not enough to fulfill the annual requirement of the country. So every year a good amount of ginger is imported in exchange of foreign currency. Like many countries diseases are the major limiting factors for ginger cultivation in Bangladesh. Among the diseases, rhizome rot is the most devastating one caused by Pythium aphanidermatum, Fusariumoxysporum, Sclerotiumrolfsiiand Ralstoniasolanacearumthroughout the world (Chauhan and Patel, 1990; Dohrooet al. 1987 and Iyer, 1987). The pathogens involved decide the nature of the damage and symptom expression .The major pathogens involved with rhizome rot are viz., species of Pythium causing soft rot, Fusariumspp. causing yellows or wilt and Ralstoniasolanacearumcausing bacterial wilt (Elliot, 2003). Basal rot caused by Sclerotiumrolfsiiwhich appears later in the season in some cases. All these pathogens are known to form complexes with nematodes leading to synergistic effect on the severity of the disease. They predispose the crops to secondary pathogens (Sarma, 1994).Loss due to rhizome rot is estimated in many countries and the main pathogens associated with rhizome rot are the fungi such as Pythium spp. and Fusariumspp, bacteria like Ralstoniasolanacearumand nematode (Elliot, 2003). It may cause losses to the extent of 50% or more due to soft rot ((Islam, 2017; BARI, 2012, 2013 and 2014; Joshi and Sharma, 1982) and sometimes total failure of the crops in the tropical regions of India (Fageriaet al, 2006); 70% rhizomeproduction is reduced due to the infection caused by Pythium spp. and Fusariumspp. in Nepal (Anon., 2012) and 5-30% losses occurred in Fiji and Australia by Pythium myriotylum(Stirlinget al., 2009). Soil, water and infected planting materials are the main source of perpetuation of these pathogens (Dohroo, 2005). Pythium spp. is able to persist in soil over decay by means of encysted zoospores, oospores and sporangia. Pythium spp also can survive in air dry muck soil for up to 12 years (Hoppe, 1966). Pythium spread via infested rhizomes (Trujillo, 1964) and as oospores surviving in debris in the soil (Dohroo, 2005). Infection started from contaminated planting materials, saprophytically living fungus in the soil or on trash of previous ginger crops. The soft rot pathogen generally have quite a wide host range and can survive on other host plants so this makes it difficult to control in the field. In the past Pythium aphanidermatumwas known as the sole causal agent of rhizome rot of ginger and was successfully controlled by the technology developed by Plant Pathology Division, BARI. But during the last few years that technology was not working well and it was noticed that some other fungal and bacterial association were involved with such rot. As the crop is cash crop so it is prime need to identify the causal agents of rhizome rot through intensive survey which will help to modify the existing technology for managing the disease in future. The present research work was undertaken to determine the incidence of rhizome rot of ginger and its causal agents.Bangladesh J. Agril. Res. 44(3): 569-576, September 2019

Downy mildew, caused by Plasmopara viticola, is a highly destructive disease of grapevine. In recent decades, European breeding programs for disease resistance have led to the creation of new cultivars resistant to downy mildew. This resistance limits mycelium growth and the sporulation of P. viticola, thereby decreasing disease epidemics due to the asexual reproduction of the pathogen. However, we still lack biological insight into the effects of partial host resistance on pathogen survival during the sexual stage of its lifecycle. We present here the results of a two-year experiment in which we assessed the production of sexual forms of P. viticola (oospores) and the success of subsequent infections on resistant cultivars with the Rpv1 and Rpv3 loci. We found that disease resistance had little effect on the sexual cycle of the pathogen. Plasmopara viticola produced four times more oospores on susceptible Vitis vinifera cultivars than on disease-resistant cultivars. Macrosporangia resulting from the germination of oospores were found on both resistant and susceptible genotypes, and were produced in surprisingly large numbers on some Rpv1 genotypes. Rates of primary contamination due to macrosporangia were lower on Rpv3 than on conventional cultivars, but higher on Rpv1 genotypes. Thus, P. viticola can complete its lifecycle (both asexual and sexual phases) on partially resistant grapevine cultivars. The maintenance of downy mildew populations from one year to the next presents a significant challenge to the sustainability of genetic resistance in grapevine.

Impatiens downy mildew is caused by Plasmopara obducens, a pathogen known in the United States for over a hundred years, but newly attacking ornamental Impatiens walleriana in production and in the landscape. Little is known about the life cycle of P. obducens; thus, in this study an attempt was made to determine whether the pathogen is homothallic or heterothallic. Fourteen single-sporangium isolates and three single-zoospore isolates were used in single and dual inoculations of stem tissue to see whether the pathogen was homothallic or heterothallic; all isolates tested were able to produce oospores when inoculated singly, suggesting homothally. Cold treatment at 0 C for at least 1 mo induced oospores to germinate and produce primary sporangia. Inoculation of plant tissue with germinating oospores resulted in infection. Other incubation temperatures (-10, 10, and 20 C) did not induce germination, but fluctuating temperatures (between -10 and 0 C, or 0 and 10 C) induced some germination. Spores incubated at -10 C had significantly thicker walls than spores incubated at other temperatures. Evidence suggests that oospores can serve as an overwintering stage.

Potato crop losses can be substantial when conditions for late blight (Phytophthora infestans) development and spread are favourable. In this study, drivers of differences between the P. infestans population structures in Latvia and Lithuania, two neighbouring countries with similar potato-growing traditions, were investigated. Genotypes of P. infestans and population genetic diversity were analysed using a 12-plex simple sequence repeat (SSR) marker assay. High genetic diversity was demonstrated in both populations, with population diversity being higher in Latvia. It would appear that local populations established from soilborne oospores early in the season are well adapted to the conditions in the region. However, somewhat greater spread and survival of local clones was detected in Lithuania, suggesting that potato cropping there is more vulnerable to clonal invasion than in Latvia. For effective disease management, current strategies should be adjusted according to the specific pathogen populations in the region, considering the reproduction and survival of the pathogen. Potato growers should implement late blight preventive measures such as longer field rotation to prevent oospore infections, especially in Latvia, and should use more disease resistant cultivars and high-quality seed potatoes.

During routine surveys of Pinus radiata plantations in the Nelson region, New Zealand, a Phytophthora species was isolated in association with bleeding stem cankers and rhizosphere soil. This isolate grew more slowly than other Phytophthora species associated with P. radiata in New Zealand, and was morphologically similar to isolates of Phytophthora cactorum previously associated with horticulture production, and isolates that were morphologically identified as P. cactorum from P. radiata in Nelson since the 1970s. Phylogenetic analyses of the ITS, cox1, and s-tubulin _F1A and s-tubulin_F2A regions confirmed this to be a new species closely related to P. hedraiandra in Clade 1. The new species is described here as Phytophthora aleatoria sp. nov. It produces partially caducous, papillate, ovoid to rarely globose or limoniform sporangia, markedly aplerotic oogonia forming thin-walled oospores, and paragynous (mainly) or amphigynous antheridia on some cultures. To date, this species has been confirmed on P. radiata in New Zealand across a range of sites within the North Canterbury, Nelson, Hawkes Bay, Taupo and the Bay of Plenty regions.

Pythium oligandrum is one of the best known fungi in biological control of many fungal plant diseases. The objective of this study was conducted to highlight the possibility of using cultural filtrate of this fungus in inhibiting the growth of reproducting units of the pathogenic Pythium aphanidermatum and P. diclinum. Two isolates of each fungus were studied. Ability of P. oligandrum to grow and develop on V-8 liquid medium was tested in order to study its effect on mycelium growth, zoospore and oospore production of each of the pathogenic pythia of P. aphanidermatum and P. diclinum. Cultural filtrate of P. oligandrum (quarter and half concentration) significantly inhibited mycelial growth and the production of zoospores and oospores of the pathogenic tested fungi. The rate of inhibition in all treatments reached more than 90% in case of the use of half concentration of the cultural filtrate of P. oligandrum. The study will provide information for the use of cultural filtrate of potential selected isolates of P. oligandrum in biological control of some fungal diseases especially in aquaculture. The outcomes of this research can be easily applied in aquatic farming but need further work on different types of pathogenic fungi.

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

Phytophthora capsici is one of the oomycetes that affects protected pepper crops in different agroclimatic areas of Spain. Currently, environmentally friendly strategies such as biodisinfestation for plant disease control have become increasingly popular. In this study, the effect of released gases during biodisinfestation with a fresh manures mixture amendment on P. capsici oospore viability was determined. A biodisinfestation trial was performed in a greenhouse located in northern Spain (Biscay), with a mixture of fresh sheep (2 kg m−2) and dry poultry manures (0.5 kg m−2) followed by soil sealing with a transparent polyethylene plastic film for 21 days (onset June 15th). Gases were sampled from the aerial cavity of biodisinfested plots at different days after soil sealing (0–1–2–3–4–7–9–11, and 14 days). Vacutainer tubes were incubated at 20°C with oospores of P. capsici that were previously placed under vacuum and refilled with extracted gases. Treatments assayed were gases from different sampling times (0–1–2–3–4–7–9–11–14 days, and succession of days 1–2–3–4–7–9–11–14) combined with different exposure times (7–14–21 days) at 20°C in the laboratory. Control treatments were included: air-tubes and vacuum-tubes. An additional reference treatment under real field conditions was also considered: buried oospores at 15 cm depth in the biodisinfested plots. Oospore viability was determined with the plasmolysis method. The most effective treatment was the succession of gases collected during all sampling days. The significant but slight reduction in oospore viability by exposure to the different gas treatments was consistent with the low dose of applied amendment and the low soil temperature registered at 15 cm depth during soil biodisinfestation (>25°C−100% time, >35°C−23%, >40°C−3%). The above circumstances might have generated a small quantity of gases with low impact on oospore viability. The biodisinfested soil at 15 cm depth reference treatment showed the lowest oospore viability in all the exposure times assayed. The overlap of thermal and higher biofumigation effects in this treatment could likely be responsible for its greater efficacy. A disinfectant effect purely attributable to released gases throughout biodisinfestation has been demonstrated. We believe that our research will serve as a base for future application in agro-environments with reduced thermal inactivation effects.

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum., Author summary Oomycetes are a class of eukaryotic microorganisms with life cycles and growth habits similar to filamentous fungi, but are not true fungi. Although sexual reproduction, which produce oospores, is an essential stage of life cycle, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. In this study, we developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium ultimum, a notorious oomycete pathogen that is responsible for a variety of diseases in a broad range of plant species. We further identified the Puf family RNA-binding protein PuM90 and the flavodoxin-like protein PuFLP as major functional factors involved in P. ultimum oospore formation. We proposed a new model that PuM90 acts as a stage-specific post-transcriptional regulator by specifically binding to the 3′-UTR of PuFLP and then repressing PuFLP mRNA level. This study describes new technologies and data that will help to elucidate sexual reproduction and post-transcriptional regulation in oomycetes.

Soybean (Glycine max) is a major source of edible oil and protein. A novel species of the genus Pythium, Pythium huanghuaiense, isolated from soybean seedlings in China, is described and illustrated on the basis of morphological characters and molecular evidence. Pythium huanghuaiense sp. nov. is closely related to species of the genus Pythium in clade F, as evidenced by the presence of hyphal swellings and its relatively rapid morphological growth. However, it differs by having relatively small sporangia and plerotic or nearly plerotic and thin-walled oospores. A pathogenicity test confirmed the newly-identified species as a pathogen of soybean.

Actinomycetes isolated from carrot rhizosphere were screened for their in vitro and in vivo antagonism to Pythium coloration Vaartaja, a causal agent of cavity-spot disease of carrots (Daucus carata L.). Forty-five streptomycete and non-streptomycete actinomycete isolates were screened for in vitro antagonism in a carrot bioassay. Of these, seven which reduced or prevented lesion formation were identified using cultural, morphological, physiological, biochemical and cell wall characteristics as Streptomyces janthimts, S. cinerochromogenes, Streptoverticilium netropsis, Actinomadura rubra, Actinoplanes philippinensis, Muromonospora carbonaceae, and Streptosporangium albidum. All seven isolates tested produced non-volatile antifungal metabolites, but failed to produce inhibitory volatile compounds. Actinoplanef philippincnsis and M. carbonacea grew epiphytically on the hyphae and oospores of P. eoloratum. The external surface of the oospores of the pathogen was heavily colonized by both hyperparasites, their hyphae were found to coil tightly around the oospore wall, and frequently caused cytoplasmic collapse of oospores. Sporangia of A. philippinensis were often seen to emerge from the colonized hyphae and oospores of P. eoloratum. None of the other actinomycete isolates showed hyperparasitism. All seven isolates significantly reduced the incidence of cavity spot in soil artificially infested with the pathogen in the glasshouse. Streptomyces janlhinus and Stfepto. albidum were the most effective in reducing the disease in inoculated plants. In addition, all the actinomycetes species except Ac. rubra and M. carbonacea, in the presence or absence of the pathogen, significantly (P < 0.05) increased mean fresh root weight compared to the treatment which included P. thorium only. This study shows that these actinomycetes have considerable potential for future use as biocontrol agents of cavity spot under natural field conditions. This is the first report of cavity-spot disease of carrots being controlled by microbial antagonists, and is the first report of non-streptomycete actinomycetes to control a Pythium disease.

Phytophthoraspecies cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species,P. agathidicida, infects and ultimately kills the treasured kauri trees that are endemic to New Zealand. Currently there are few options for controlling or treatingP. agathidicida. In this study, we sought to assess the toxicity of the oomycide oxathiapiprolin against several lifecycle stages of two geographically distinctP. agathidicidaisolates. Half maximal effective concentration (EC50) values were determined to be approximately 0.1 ng/ml for inhibiting mycelial growth, indicating thatP. agathidicidamycelia are more sensitive to oxathiapiprolin than those from most otherPhytophthoraspecies that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50= 2-9 ng/ml for the two isolates) and oospores (complete inhibition at 100 ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.