Oospore Formation Research Articles

The growth‐promoting effects of Bacillus amyloliquefaciens W82T‐44 on soybean and its biocontrol potential against soybean Phytophthora root rot

AbstractThe most devastating soilborne disease of soybean is Phytophthora root rot (PRR) caused by Phytophthora sojae. Biological control has emerged as an effective method of reducing soilborne diseases. The present study isolated the bacterial strain W82T‐44 from the rhizosphere soil of a resistant soybean variety Williams 82. This strain was identified as Bacillus amyloliquefaciens and exhibited a 92.4% inhibition of mycelial growth of P. sojae. W82T‐44 produced cellulase, siderophore and protease and significantly promoted soybean growth. W82T‐44 treatment, compared to the negative control, was found to significantly (p < 0.05) increase soybean plant height (22.34 vs. 18.47 cm), fresh weight of the whole plant (2.57 vs. 2.05 g), dry weight (0.43 vs. 0.32 g), total root length (266.80 vs. 182.53 cm), root area (27.10 vs. 17.84 cm2) and root volume (1.88 vs. 1.16 cm3). The fermentation filtrate of W82T‐44 had significant inhibitory effects on mycelial growth, oospore formation and cyst germination in P. sojae and additionally promoted zoospore encystment. W82T‐44 possessed genes involved in lipopeptide synthesis, including bacilysin, surfactin, fengycin and bacillomycin. Moreover, it significantly upregulated pathogenesis‐related genes (β‐1,3‐glucanase, nonexpressor of pathogenesis‐related gene 1, chitinase) and genes encoding defence enzymes (polyphenol oxidase, phenylalanine ammonia‐lyase, peroxidase) in soybean roots (p < 0.05). The PRR disease index of soybean treated with the W82T‐44 strain was significantly lower (11.67) than the negative control (40.00; p < 0.05). The present study indicates the potential of B. amyloliquefaciens W82T‐44 as a biological control agent against PRR from various perspectives.

The ankyrin repeat-containing protein PlANK1 mediates mycelial growth, oospore development, and virulence in Peronophythora litchii

Litchi downy blight, caused by Peronophythora litchii, is one of the most serious diseases in major litchi-producing regions worldwide. The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs found in all kingdoms of life proteins. ANK-containing proteins have been demonstrated to regulate various biological processes in animals, plants, and fungi. However, their functions in phytopathogenic oomycetes remain unknown. Here, we identified 284 non-redundant genes that encode ANK-containing proteins in P. litchii and classified them into 11 subfamilies. Among them, PlANK1 was found to be highly up-regulated in oospores and from zoospores to the infection process. Loss of PlANK1 in P. litchii resulted in impaired mycelial growth and cyst germination, accelerated zoospore encystment, and increased sensitivity to hyperosmotic stresses and Congo red. Furthermore, PlANK1 deletion mutants were defective in oospore formation and development. Inoculation assays showed that the absence of PlANK1 severely diminished the pathogen’s virulence on litchi leaf and fruit. Through transcriptome analysis and nitrogen source utilization assays, we demonstrated that PlANK1 modulates the pathogen’s nitrogen metabolism. Altogether, our findings indicate that PlANK1 is a key regulator of sexual and asexual development, and virulence in P. litchii.

A major facilitator superfamily transporter PlMFS1 contributes to growth, oosporogenesis, and pathogenesis of Peronophythora litchii

Major facilitator superfamily (MFS) transporters are secondary active membrane transporters that play an important role in solute interchange and energy metabolism. Peronophythora litchii causes the most destructive disease on lichi, litchi downy blight. PlM90 was reported as a key oosporogenesis regulator. Here, we identified an MFS transporter gene PlMFS1, which is up-regulated during oospore formation at the late infection stage, while down-regulated in the PlM90 mutant. To investigate PlMFS1 function, we generated PlMFS1 knockout mutants using CRISPR/Cas9-mediated genome editing technology. Compared with the wild-type strain SHS3, PlMFS1 deletion impaired mycelium growth, zoospore release, oospore production and pathogenicity. Furthermore, PlMFS1 deletion significantly affected P. litchii utilization of fructose, lactose and maltose, and may be the PlMFS1 mechanism involved in mycelial growth. PlMFS1 gene deletion also led to deceased laccase activity, laccase-encoding gene downregulation and impaired P. litchii pathogenicity. To our knowledge, this is the first report of an MFS transporter involved in sugar utilization, sexual reproduction, asexual reproduction and pathogenesis in oomycetes.

Selective Isolation and Characterization of Phytophthora infestans from Potatoes Using Rye Agar Media in India

Phytophthora infestans is a pathogen that causes late blight, a major disease of potatoes. The isolation of P. infestans from infected potato plants using agar media has been challenging. This study investigated the use of Rye A and Rye B agar media for the isolation of P. infestans from infected potato tubers collected from the Nilgiris district of Tamil Nadu during 2022. The media were evaluated for hyphal growth, sporangial production, oospore formation, and long-term storage of P. infestans. Phenotypic diagnosis based on cultural and morphological characteristics confirmed the identity of P. infestans. The results were confirmed by a molecular identification test using primers specific to P. infestans. Pathogenicity tests were carried out to assess the virulence of the isolates. This study provides a useful protocol for the selective isolation and characterization of P. infestans, the potato late blight pathogen.

Phytophthora capsici: Recent Progress on Fundamental Biology and Disease Management 100 Years After Its Description.

Phytophthora capsici is a destructive oomycete pathogen of vegetable, ornamental, and tropical crops. First described by L.H. Leonian in 1922 as a pathogen of pepper in New Mexico, USA, P. capsici is now widespread in temperate and tropical countries alike. Phytophthora capsici is notorious for its capability to evade disease management strategies. High genetic diversity allows P. capsici populations to overcome fungicides and host resistance, the formation of oospores results in long-term persistence in soils, zoospore differentiation in the presence of water increases epidemic potential, and a broad host range maximizes economic losses and limits the effectiveness of crop rotation. The severity of disease caused by P. capsici and management challenges have led to numerous research efforts in the past 100 years. Here, we discuss recent findings regarding the biology, genetic diversity, disease management, fungicide resistance, host resistance, genomics, and effector biology of P. capsici.

Isoflavone phytoalexins in root exudates participate in mediating the resistance of common bean Phaseolus vulgaris to Phytophthora sojae

AbstractCommon bean (Phaseolus vulgaris) cannot be infected naturally by Phytophthora sojae but produces a hypersensitive response (HR) under P. sojae inoculation. The goal of this study was to determine the roles of isoflavone phytoalexins in common bean root exudates in the resistance of common bean to P. sojae. The concentrations of kievitone, phaseollin and phaseollidin in the root exudates of common bean and their effects on P. sojae were measured. The transcriptomes of P. sojae responding to the phytoalexins were analysed. The three phytoalexins were included in the root exudates of three common bean cultivars at different concentrations and inhibited mycelial growth, oospore formation, zoospore chemotaxis and infection efficiency of P. sojae. The concentrations of the three phytoalexins in root exudates of three common bean cultivars were very low but increased significantly after inoculation with P. sojae. Kievitone probably inhibits P. sojae by promoting accumulation of abnormal transcripts, destroying mitochondria, inhibiting synthesis of ATP and protein, and reducing cell invasion and migration, making cells more sensitive to oxidative stress and slowing down proliferation and even apoptosis; phaseollin probably inhibits P. sojae by promoting the transcription level of some key genes in P. sojae that are associated with a reduction in migration and cell membrane damage; and phaseollidin probably inhibits P. sojae by promoting cell apoptosis. Our results suggest that kievitone, phaseollin and phaseollidin are involved in mediating the resistance of common bean to P. sojae through different mechanisms.

Signal and regulatory mechanisms involved in spore development of Phytophthora and Peronophythora.

Oomycetes cause hundreds of destructive plant diseases, threatening agricultural production and food security. These fungus-like eukaryotes show multiple sporulation pattern including the production of sporangium, zoospore, chlamydospore and oospore, which are critical for their survival, dispersal and infection on hosts. Recently, genomic and genetic technologies have greatly promoted the study of molecular mechanism of sporulation in the genus Phytophthora and Peronophythora. In this paper, we characterize the types of asexual and sexual spores and review latest progress of these two genera. We summarize the genes encoding G protein, mitogen-activated protein kinase (MAPK) cascade, transcription factors, RNA-binding protein, autophagy-related proteins and so on, which function in the processes of sporangium production and cleavage, zoospore behaviors and oospore formation. Meanwhile, various molecular, chemical and electrical stimuli in zoospore behaviors are also discussed. Finally, with the molecular mechanism of sporulation in Phytophthora and Peronophythora is gradually being revealed, we propose some thoughts for the further research and provide the alternative strategy for plant protection against phytopathogenic oomycetes.

Field Occurrence and Overwintering of Oospores of Pseudoperonospora cubensis in the Southeastern United States.

In the United States, the cucurbit downy mildew pathogen, Pseudoperonospora cubensis, has been shown to form oospores under laboratory conditions, but there are no reports on the formation of oospores in naturally infected cucurbit plants in the field. This study investigated the occurrence of oospores in naturally infected leaves from cucurbit fields in North Carolina and South Carolina from 2018 to 2020. Oospore viability and survival was also determined outdoors during the winter in North Carolina during this study period using soil containing leaves infested with oospores. About 5% of 1,658 naturally infected cucumber and cantaloupe leaves sampled during the study had oospores, with a mean density of 585 oospores per cm2 of infected leaf tissue. Absolute oospore viability, as assessed using the plasmolysis method, declined linearly (slope = -0.27; P < 0.0001) over the 6-month exposure period from 67.8% in November to 19.3% in May. Other variables being equal, the decrease in oospore viability was significantly affected by soil temperature (b = -0.03 to -0.05; P < 0.0001) and number of rainy days (b = 21.6 to 40.46; P < 0.05), while the effects of soil moisture on oospore viability were less clear. About 20% of the oospores exposed to outdoor conditions at the end the study period were putatively viable and deemed potentially infective. However, these putatively viable oospores failed to germinate or initiate disease when inoculated onto cucumber or cantaloupe leaves. These results indicate that oospores might require some unrecognized stimuli or physiological factors to initiate germination and infection. Nonetheless, viability of oospores at the end of the winter season suggests that once exposed to the right conditions that stimulate germination, these oospores could potentially serve as a primary inoculum source in the southeastern United States where winter temperatures are cold enough to kill cucurbits plants.

Changes in biochemistry and cellular ultrastructure support different resistance mechanisms to Phytophthora sojae in nonhost common bean and host soybean

AbstractPhytophthora root and stem rot of soybean caused by Phytophthora sojae is a destructive disease affecting soybean production worldwide. In nature, soybean is the only economically important cultivated host of P. sojae. The aim of this study was to explain different resistance mechanisms to P. sojae in nonhost common bean and host soybean as a basis for the control of Phytophthora root and stem rot of soybean via nonhost resistance. Observations and measurements of disease resistance‐related variables showed slight differences in structural and biochemical resistance mechanisms between common bean and soybean. P. sojae infection induced a stronger hypersensitive response in nonhost common bean than in host resistant soybean. Moreover, phytoalexin phaseollidin synthesis‐related vestitone reductase gene was extremely highly up‐regulated, and phytoalexin glyceollin synthesis‐related isoflavone reductase gene was slightly less up‐regulated in common bean than in soybean, which resulted in a higher level of phaseollidin and a lower level of glyceollin in common bean. Phaseollidin had stronger inhibitory effects on mycelial growth and oospore formation of P. sojae than glyceollin, and more cell wall depositions and callose accumulated in common bean, which are probably related to the stronger resistance of nonhost common bean to P. sojae.

Specific interaction of an RNA-binding protein with the 3'-UTR of its target mRNA is critical to oomycete sexual reproduction.

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.

First report of oospore formation in Plasmopara viticola, the causal agent of grapevine downy mildew, in highland regions of southern Brazil

AbstractThis work evaluates the formation of oospores of Plasmopara viticola, the causal agent of grape downy mildew (DM), in highland regions in southern Brazil. Leaves of susceptible and resistant grape genotypes naturally infected with the pathogen were collected in the autumn of 2017, 2018, and 2019 from vineyards located in the highlands of Santa Catarina state. Leaf tissues were evaluated by light microscopy and scanning electron microscopy. Oospores of P. viticola were identified in both susceptible and resistant host genotypes. They were concentrated in the central regions of the DM lesions, close to the leaf veins, and exhibited a rounded shape, yellowish colour, thick wall, and a diameter ranging from 16.28 to 49.15 µm. The formation of oospores is strong evidence that sexual reproduction is occurring in P. viticola in the climatic conditions of the highlands of southern Brazil. Sexual reproduction contributes to the maximization of genetic diversity via meiosis. Populations with high genetic variability are more likely to break resistance mechanisms conferred by resistance genes and to develop resistance to fungicides applied for disease control. To our knowledge, this is the first scientific study to prove the formation of P. viticola oospores in Brazil. The results presented provide a solid basis for further studies on sexual recombination in P. viticola. Genetic improvement programmes for grapevines, disease management methods, and disease prediction models need to consider the sexual reproduction of this pathogen, otherwise their effectiveness may be compromised.

Effect of light and dark on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis

AbstractDisease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant–oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation, and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hr. In contrast, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development, and oospore formation, whereas exposure to constant light stimulated these three stages of development. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.

Dominance of Mating Type A1 and Indication of Epigenetic Effects During Early Stages of Mating in Phytophthora infestans.

The potato late blight pathogen Phytophthora infestans has both an asexual and a sexual mode of reproduction. In Scandinavia, the pathogen is reproducing sexually on a regular basis, whereas clonal lineages dominate in other geographical regions. This study aimed at elucidating events or key genes underlying this difference in sexual behavior. First, the transcriptomes of eight strains, known as either clonal or sexual, were compared during early stages of mating. Principal component analysis (PCA) divided the samples in two clusters A and B and a clear grouping of the mating samples together with the A1 mating type parents was observed. Induction of genes encoding DNA adenine N6-methylation (6mA) methyl-transferases clearly showed a bias toward the cluster A. In contrast, the Avrblb2 effector gene family was highly induced in most of the mating samples and was associated with cluster B in the PCA, similarly to genes coding for acetyl-transferases, which play an important role in RXLR modification prior to secretion. Avrblb2 knock-down strains displayed a reduction in virulence and oospore formation, suggesting a role during the mating process. In conclusion, a number of gene candidates important for the reproductive processes were revealed. The results suggest a possible epigenetic influence and involvement of specific RXLR effectors in mating-related processes.

Variabilidad morfológica y sensibilidad de Phytophthora capsici causando marchitez en chile pimiento morrón en Chihuahua, México

A nivel mundial, México es el principal exportador del chile pimiento morrón (&lt;em&gt;Capsicum annuum&lt;/em&gt;). El cultivo es afectado por diversas enfermedades, siendo la marchitez del chile ocasionada por &lt;em&gt;Phytophthora capsici&lt;/em&gt; una de las más importantes. Este fitopatógeno posee alta variabilidad en virulencia y sensibilidad a fungicidas. No existen en México estudios sobre poblaciones de &lt;em&gt;P. capsici &lt;/em&gt; para pimiento morrón. El objetivo de este trabajo fue determinar la diversidad de aislados de &lt;em&gt;P. capsici&lt;/em&gt; obtenidos de plantas de pimiento morrón con marchitez en Delicias, Chihuahua, México, mediante los marcadores fenotípicos: patrón de crecimiento de la colonia, tipo de compatibilidad, virulencia y sensibilidad a fungicidas. Los tipos de colonia fueron: estrellado (74%), ligeramente petaloide (14%) y radial (12%). Se detectaron aislados sensibles (53%), con sensibilidad intermedia (42%) e insensibles (5%) a mefenoxam. El 86.6% de los aislados inoculados fueron altamente virulentos, produjeron síntomas cuatro días después de la inoculación. Se detectaron los dos tipos de compatibilidad (TC) A1 y A2. Se formaron oosporas &lt;em&gt;in planta&lt;/em&gt; al inocular los dos TC detectados en una misma planta. Los resultados sugieren que existe reproducción sexual en campo y como consecuencia variabilidad entre los aislados de &lt;em&gt;P. capsici&lt;/em&gt;, responsables de la marchitez en cultivos de chile pimiento morrón en Delicias, Chihuahua, México.

Karakteristik morfologi dan sebaran tipe kawin Phytophthora capsici asal lada di Pulau Jawa

Morphology characters and mating types distribution of Phytophthora capsici from black pepper in Java IslandPepper (Piper nigrum) is one of the most important spice crops in Indonesia. Recently its production declining due to infection of foot rot disease caused by Phytophthora capsici. This pathogen has two different mating types, namely A1 and A2, in which the presence of opposite two mating types is important for sexual reproduction and formation of oospores. The movement of pepper seedling from one area to another is highly facilitated alteration of mating type distribution map of P. capsici. The objectives of this research were to determine the morphological characteristics and the spread of mating types of P. capsici in Java. Morphology characters of P. capsici isolates were indicated by variation in sporangial size and shape, as well as types of colony appearance. The length (l) and width (w) of sporangium were in the range of 15.1–76.2 µm and 9.8–44.8 µm, respectively; while the l/w ratio was 1.12–2.27. Mating type assay showed that A2 type was more dominantly found than A1 type. This study found two different mating types present in the same area, i.e. Regency of Pacitan (East Java) and Regency of Sleman (Special Region of Yogyakarta). The findings of this research suggested that it is required more strict control strategy on the mobilization of black pepper seedling particularly in the area where the certain mating type is not found yet so that the emergence of new more virulent genotype of pathogen can be prevented.

Efecto in vitro de fosfito de potasio sobre Athelia rolfsii y Pythium aphanidermatum

El objetivo de este estudio fue determinar el efecto in vitro del fosfito de potasio sobre el crecimiento radial del micelio, la producción de biomasa y la producción de esclerocios de Athelia rolfsii y de oosporas de Pythium aphanidermatum. Dosis de 0.05, 0.1, 0.15, 0.2, 0.25 y 0.3 mL L-1 de fosfito de potasio y un testigo absoluto (sin el fosfito de potasio) fueron evaluados utilizando medio de cultivo PDA y agua destilada. El crecimiento radial de micelio disminuyó debido al fosfito de potasio, por lo que al final de la evaluación y comparándolo con el control, el crecimiento radial de P. aphanidermatum disminuyó de 16.9 a 53.2% y de 15 a 21.3% para A. rolfsii. A las 96 h después de la siembra la producción de biomasa por P. aphanidermatum y A. rolfsii, disminuyó de 16.9 a 53.2% y 58.3 a 63.4%, respectivamente. Después de 72 h de la siembra, no se observó la formación de oosporas de P. aphanidermatum sobre agua destilada con fosfito de potasio, lo que sí ocurrió en el control después de 24 h. A los 22 días después de la siembra, A. rolfsii produjo un promedio de 30.2 esclerocios, mientras que en PDA con fosfito de potasio no formó esclerocios. Esto indica que el fosfito de potasio es una sustancia eficaz para reducir el crecimiento del micelio, la producción de biomasa, e inhibe la formación de oosporas de P. aphanidermatum y de esclerocios de A. rolfsii.

Mating types of Phytophthora colocasiae on the island of Upolu, Samoa

Taro leaf blight (TLB) caused by Phytophthora colocasiae is a damaging disease that destroyed Samoa’s taro industry following its introduction in 1993. The aim of this study was to determine the occurrence of the A1 and A2 mating types of P. colocasiae for a more comprehensive understanding of the risk the pathogen poses for the future of the taro industry in Samoa. In September 2015, 54 isolates of P. colocasiae were collected from taro leaf blight lesions from 13 farms around the island of Upolu, Samoa. The mating types of each isolate was determined by observation of oospore formation when paired with tester isolates of Phytophthora nicotianae of known mating types (A1 or A2). Fifty isolates were found to be A2 mating type and four did not form oospores with either mating type. No A1 or self-fertile isolates were found. These results suggest that the A1 mating type has not been introduced to the island of Upolu, preventing the formation of oospores between compatible mating types of P. colocasiae and lessening the risk of new and potentially more threatening genotypes of the pathogen from emerging through genetic recombination.&#x0D; Keywords taro leaf blight, Colocasia esculenta, taro, sexual reproduction

Baseline Sensitivities of New Fungicides and Their Toxicity to Selected Life Stages of Phytophthora Species from Citrus in California.

Phytophthora citrophthora, P. syringae, P. nicotianae, and P. hibernalis are important pathogens of citrus in California but few chemical treatments are currently available. In vitro toxicities of four new fungicides to isolates of Phytophthora spp. from California were determined. Mean effective concentration values to inhibit mycelial growth by 50% for ethaboxam, fluopicolide, mandipropamid, oxathiapiprolin, and mefenoxam were 0.068, 0.04, 0.004, 0.0003, and 0.039 µg/ml, respectively, for 62 isolates of P. citrophthora; 0.005, 0.045, 0.003, 0.0001, and 0.008 µg/ml, respectively, for 71 isolates of P. syringae; 0.016, 0.057, 0.005, 0.0005, and 0.183 µg/ml, respectively, for 31 isolates of P. nicotianae; and 0.030, 0.018, 0.005, <0.0003, and ≤0.001 µg/ml, respectively, for two isolates of P. hibernalis. Mean values for ≥90% inhibition of sporangia formation of four isolates of P. citrophthora were 0.1, 0.28, 0.026, 0.005, and 55 µg/ml for ethaboxam, fluopicolide, mandipropamid, oxathiapiprolin, and mefenoxam, respectively. Zoospore cyst germination of P. citrophthora was most inhibited by oxathiapiprolin and mandipropamid. Chlamydospore formation of P. nicotianae was most sensitive to oxathiapiprolin, with a mean ≥90% reduction (EC>90) of 0.002 µg/ml, moderately sensitive to mandipropamid (EC>90 = 0.2 µg/ml) and mefenoxam (EC>90 = 0.6 µg/ml), and least sensitive to ethaboxam and fluopicolide (EC>90 = 1 µg/ml). Oospore formation of P. nicotianae was inhibited by ≥90% using oxathiapiprolin at 0.0004 µg/ml, mandipropamid at 0.02 µg/ml, ethaboxam at 0.1 µg/ml, or fluopicolide at 0.4 µg/ml, whereas 62% inhibition was obtained by mefenoxam at 40 µg/ml.

The Loricrin-Like Protein (LLP) of Phytophthora infestans Is Required for Oospore Formation and Plant Infection.

Loricrin-like protein (LLP) is characterized by a high content of glycine residues and is a major component of plant cell wall. Here, we identified a Phytophthora infestans ortholog of plant LLP, named PiLLP. In P. infestans, PiLLP is strongly expressed in asexual and sexual developmental stages, including in sporangia, zoospores and germinating cysts, and during oospore formation, as well as in the early stages of infection and during hydrogen peroxide stress. Compared with the wild type, the PiLLP-silenced transformants were defective in oospore formation, had slower colony expansion rates, produced less sporangia with lower germination and zoospore-release rates, and were more sensitive to hydrogen peroxide. Moreover, Nile red staining, and PiLLP-red fluorescent protein fusions indicated that PiLLP is involved in oogonia formation. The silenced transformants also had severely diminished virulence levels that could be partially restored with diphenyleneiodium treatments. The analysis of catalase activity showed a decrease of catalase activity in silenced transformants. Thus, PiLLP is important for sexual and asexual reproduction, and is required for oxidative stress tolerance and plant infection.

Occurrence and Distribution of Mating Types of Pseudoperonospora cubensis in the United States.

During the past two decades, a resurgence of cucurbit downy mildew has occurred around the world, resulting in severe disease epidemics. In the United States, resurgence of the disease occurred in 2004 and several hypotheses, including introduction of a new genetic recombinant or pathotype of the pathogen, have been suggested as potential causes for this resurgence. Occurrence and distribution of mating types of Pseudoperonospora cubensis in the United States were investigated using 40 isolates collected from cucurbits across 11 states from 2005 to 2013. Pairing of unknown isolates with known mating-type tester strains on detached leaves of cantaloupe or cucumber resulted in oospore formation 8 to 10 days after inoculation. Isolates differed in their ability to form oospores across all coinoculation pairings, with oospore numbers ranging from 280 to 1,000 oospores/cm2 of leaf tissue. Oospores were hyaline to golden-yellow, spherical, and approximately 36 μm in diameter. Of the 40 isolates tested, 24 were found to be of the A1 mating type, while 16 were of the A2 mating type. Mating type was significantly (P < 0.0001) associated with host type, whereby all isolates collected from cucumber were of the A1 mating type, while isolates from squash and watermelon were of the A2 mating type. Similarly, mating type was significantly (P = 0.0287) associated with geographical region, where isolates from northern-tier states of Michigan, New Jersey, New York, and Ohio were all A1, while isolates belonging to either A1 or A2 mating type were present in equal proportions in southern-tier states of Alabama, Florida, Georgia, North Carolina, South Carolina, and Texas. Viability assays showed that oospores were viable and, on average, approximately 40% of the oospores produced were viable as determined by the plasmolysis method. This study showed that A1 and A2 mating types of P. cubensis are present and the pathogen could potentially reproduce sexually in cucurbits within the United States. In addition, the production of viable oospores reported in this study suggests that oospores could have an important role in the biology of P. cubensis and could potentially influence the epidemiology of cucurbit downy mildew in the United States.