The Gß-like protein Bcgbl1 regulates development and pathogenicity of the gray mold Botrytis cinerea via modulating two MAP kinase signaling pathways.

The fungal Gß-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gß-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gß-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea.

Biological Control of Rapeseed Clubroot (Plasmodiophora brassicae) using the Endophytic Fungus Didymella macrostoma P2.

Didymella macrostoma P2 was isolated from rapeseed (Brassica napus), and it is an endophyte of rapeseed and an antagonist of three rapeseed pathogens, Botrytis cinerea, Leptosphaeria biglobosa and Sclerotinia sclerotiorum. However, whether or not P2 has a suppressive effect on infection of rapeseed by the clubroot pathogen Plasmodiophora brassicae remains unknown. This study was conducted to detect production of antimicrobials by P2 and to determine efficacy of the antimicrobials and P2 pycnidiospores in suppression of rapeseed clubroot. Results showed that cultural filtrates (CF) of P2 in potato dextrose broth and the substances in pycnidiospore mucilages exuded from P2 pycnidia were inhibitory to P. brassicae. In the indoor experiment, seeds of the susceptible rapeseed cultivar Zhongshuang No.9 treated with P2 CF and the P2 spore suspension (P2 SS, 1 × 107 spores/ml) reduced clubroot severity by 31% to 70% on the 30-day-old seedlings compared to the control (seeds treated with water). P2 was re-isolated from the roots of the seedlings in the treatment of P2 SS, the average isolation frequency in the healthy roots (26%) was much higher than that (5%) in the diseased roots. In the field experiment, seeds of another susceptible rapeseed cultivar Huayouza 50 (HYZ50) treated with P2 CF, P2 CE (chloroform extract of P2 CF, 30 µg/ml) and P2 SS reduced clubroot severity by 29% to 48% on 60-day-old seedlings and by 28% to 59% on adult plants (220 days old) compared to the control treatment. The three P2 treatments on HYZ50 produced significantly (P < 0.05) higher seed yield than the control treatment on this rapeseed cultivar, and they even generated seed yield similar to that produced by the resistant rapeseed cultivar Shengguang 165R in one of the two seasons. These results suggest that D. macrostoma P2 is an effective biocontrol agent against rapeseed clubroot.

Botrytis cinerea BcSSP2 protein is a late infection phase, cytotoxic effector.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcSSP2) that can also induce immune resistance in plants. BcSSP2 is a small, cysteine-rich protein without any known domains. Transient expression of BcSSP2 in leaves caused chlorosis that intensifies with time and eventually leads to death. Point mutations in eight of 10 cysteine residues abolished phytotoxicity, but residual toxic activity remained after heating treatment, suggesting contribution of unknown epitopes to protein phytotoxicity. The expression of bcssp2 was low during the first 36 h after inoculation and increased sharply upon transition to late infection stage. Deletion of bcssp2 did not cause statistically significant changes in lesions size on bean and tobacco leaves. Further analyses indicated that the phytotoxicity of BcSSP2 is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcSSP2 is an effector protein that toxifies the host cells, but is also recognized by the plant immune system.

Biological characterization of the melanin biosynthesis gene Bcscd1 in the plant pathogenic fungus Botrytis cinerea.

The gray mold fungus Botrytis cinerea produces dark-colored conidia and sclerotia due to deposition of melanin on the cell wall of these structures. However, the role of melanin biosynthesis on development and function of conidia and sclerotia have not been well elucidated in this fungus. This study disrupted the melanin biosynthesis gene Bcscd1 (for scytalone dehydratase) in the wild type B05.10, and the resulting mutants were compared with B05.10 and complementary mutants (COM) for growth and development, virulence and response to biotic/abiotic stresses. Three disruption mutants were obtained, and they did not differ from B05.10 and COM in mycelial growth rate on potato dextrose agar, however, they formed brownish conidia and scleotia deficient in melanogenesis, whereas B05.10 and COM formed grayish conidia and black sclerotia with normal melanogenesis. The disruption mutants were as aggressive as B05.10 and COM in infection of tobacco leaves. TEM observation showed that the disruption mutant ΔScd1-85 formed numerous tiny grooves in the conidial cell wall, thereby causing uneven thickness in the cell wall. In contrast, B05.10 and COM rarely formed tiny grooves in their conidial cell wall with even thickness. Moreover, the sclerotial cortex cell wall of ΔScd1-85 lost rigidity and the cells became collapsed, whereas the sclerotial cortex cell wall of B05.10 and COM appeared rigid, and the cells appeared plump in shape. The disruption mutants were more sensitive than B05.10 and COM in response to chemical stresses (H2O2, NaCl, SDS, sorbitol) for conidial germination and sclerotial survival. The sclerotia of the disruption mutants were more susceptible than the sclerotia of B05.10 and COM to infection by the mycoparasite Trichoderma koningiopsis. These results confirmed previous studies about the effect of melanin production on pathogenicity of B. cinerea, and expanded our knowledge about the role of Bcscd1 in cell wall integrity and in response to biotic and abiotic stresses.

Molecular characterization of a novel fusagravirus in the phytopathogenic fungus Streptobotrys caulophylli.

Streptobotrys caulophylli is a pathogenic fungus that causes leaf and stem blight in many plants. We report a novel fusagravirus identified in S. caulophylli strain STB-2, provisionally named "Streptobotrys caulophylli fusagravirus 1" (ScFV1). The full-length genome of ScFV1 is 9270 nucleotides (nt) long and putatively possesses two large open reading frames (ORF1 and ORF2), which are separated by an intergenic region of 955 nt. A conserved domain search revealed that ORF2 (1051 aa) encodes a putative RNA-dependent RNA polymerase (RdRp), whereas ORF1 (1404 aa) encodes a protein of unknown function. Homology searches and phylogenetic analysis of the putative RdRp suggest that ScFV1 is a new member of the proposed family "Fusagraviridae". This is the first report of a mycovirus infecting the fungus S. caulophylli.

First Report of Leptosphaeria biglobosa 'brassicae' Causing Blackleg on Brassica juncea var. multisecta in China.

Brassica juncea var. multisecta, a leafy mustard, is widely grown in China as a vegetable (Fahey 2016). In May 2018, blackleg symptoms, grayish lesions with black pycnidia, were found on stems and leaves of B. juncea var. multisecta during disease surveys in Wuhan, Hubei Province. Disease incidence was approximately 82% of plants in the surveyed fields (~1 ha in total). To determine the causal agent of the disease, twelve diseased petioles were surface-sterilized and then cultured on potato dextrose agar (PDA) at 20˚C for 5 days. Six fungal isolates (50%) were obtained. All showed fluffy white aerial mycelia on the colony surface and produced a yellow pigment in PDA. In addition, pink conidial ooze formed on top of pycnidia after 20 days of cultivation on a V8 juice agar. Pycnidia were black-brown and globose with average size of 145 × 138 µm and ranged between 78 to 240 × 71 to 220 µm, n = 50. The conidia were cylindrical, hyaline, and 5.0 × 2.1 µm (4 to 7.1 × 1.4 to 2.9 µm, n=100). These results indicated that the fungus was Leptosphaeria biglobosa rather than L. maculans, as only the former produces yellow pigment (Williams and Fitt 1999). For molecular confirmation of identify, genomic DNAs were extracted and tested through polymerase chain reaction (PCR) assay using the species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006), of which DNA samples of L. maculans isolate UK-1 (kindly provided by Dr. Yongju Huang of University of Hertfordshire) and L. biglobosa 'brassicae' isolate B2003 (Cai et al. 2014) served as controls. Moreover, the sequences coding for actin, ß-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (Vincenot et al. 2008) of isolates HYJ-1, HYJ-2 and HYJ-3 were also cloned and sequenced. All six isolates only produced a 444-bp DNA fragment, the same as isolate B2003, indicating they belonged to L. biglobosa 'brassicae', as L. maculans generates a 331-bp DNA fragment. In addition, sequences of ITS (GenBank accession no. MN814012, MN814013, MN814014), actin (MN814292, MN814293, MN814294), and ß-tubulin (MN814295, MN814296, MN814297) of isolates HYJ-1, HYJ-2 and HYJ-3 were 100% identical to the ITS (KC880981), actin (AY748949), and ß-tubulin (AY748995) of L. biglobosa 'brassicae' strains in GenBank, respectively. To determine their pathogenicity, needle-wounded cotyledons (14 days) of B. juncea var. multisecta 'K618' were inoculated with a conidial suspension (1 × 107 conidia/ml, 10 µl per site) of two isolates HYJ-1 and HYJ-3, twelve seedlings per isolate (24 cotyledons), while the control group was only treated with sterile water. All seedlings were incubated in a growth chamber (20°C, 100% relative humidity under 12 h of light/12 h of dark) for 10 days. Seedlings inoculated with conidia showed necrotic lesions, whereas control group remained asymptomatic. Two fungal isolates showing the same culture morphology to the original isolates were re-isolated from the necrotic lesions. Therefore, L. biglobosa 'brassicae' was confirmed to be the causal agent of blackleg on B. juncea var. multisecta in China. L. biglobosa 'brassicae' has been reported on many Brassica crops in China, such as B. napus (Fitt et al. 2006), B. oleracea (Zhou et al. 2019), B. juncea var. multiceps (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020). To our knowledge this is the first report of L. biglobosa 'brassicae' causing blackleg on B. juncea var. multisecta in China, and its occurrence might be a new threat to leafy mustard production of China.

First report of Leptosphaeria biglobosa 'brassicae' causing Black Leg on Brassica rapa subsp. pekinensis in China.

Chinese cabbage [Brassica rapa L. subsp. pekinensis (Lour.) Hanelt] is a major leafy vegetable crop grown in China and eastern Asia (Fordham and Hadley 2003). In December 2018, black leg symptoms were observed on of "Qingza No.3" of Chinese cabbage during harvest, Chibi (29°46'37.38''N, 114°05'6.88''E), Hubei, China. Symptoms were first noted in late Nov. as black spots on leaf petioles and basal stems. Then, black spots enlarged as oval or irregular-shaped grayish lesions. Finally, lesions enlarged and coalesced with black pycnidia were observed, and some diseased leaves became blighted. The disease incidence was about 80% in three fields surveyed (~2 ha in total). Diseased plant tissues were surface-sterilized, and incubated on potato dextrose agar (PDA) plates at 20°C for 4 days. Three fungal isolates, namely EP9-19, EP9-22 and EP9-26, were obtained from five of the diseased samples; all produced fluffy, white aerial mycelia and a yellow pigment on PDA. After 14 days, black-brown and globose pycnidia were produced, approximately 150 µm in diameter (n = 50). In addition, pink pycnidiospore ooze was observed on the top of pycnidium after 20-day culturing on a V8-juice (20%) agar. Conidia were cylindrical and hyaline, with the mean size of 4.6 × 2.7 µm (n = 50). Two fungal species have been reported to cause blackleg on Brassica crops (Williams and Fitt 1999), i.e. Leptosphaeria maculans and L. biglobosa. The former is much more destructive, but is not present in China. These isolates had morphological characteristics matching those of L. biglobosa (Williams and Fitt 1999). The genomic DNA of isolate EP9-22 was extracted and sequenced for its actin, ß-tubulin and the internal transcribed spacer (ITS) region of ribosomal DNA as described by Vincenot et al. (2008). Sequences of ITS (GenBank accession no. MN238766), actin (MN242213) and ß-tubulin (MN242214) for isolate EP9-22 showed 100%, 99.67%, and 97.93% identity to the corresponding regions of L. biglobosa 'brassicae' strain IBCN89 (Vincenot et al. 2008). In addition, the phylogenetic analysis also indicated that isolate EP9-22 belonged to L. biglobosa 'brassicae'. The pathogenicity test was performed according to established protocols (Balesdent et al., 2005). Cotyledons of the 15-day-old Chinese cabbage seedlings (cultivars Xiaoza No.55 and Hualiangzao No.5) were wound inoculated with 10 µl pycnidiospore suspension (1 × 107 conidia/ml) of the three isolates, with 20 cotyledons per isolate, respectively, and 20 cotyledons wound inoculated with sterile water served as a control group. The treated seedlings were maintained at 20°C and 100% relative humidity with a 12-h photoperiod. The experiment was repeated twice. At 7 days after inoculation, necrotic lesions became visible surrounding inoculation sites for the three isolates, while the control group remained healthy. Fungal isolates showing a similar colony morphology to the originals were re-isolated from ten diseased cotyledons but not from the control cotyledons. Based on these results, L. biglobosa 'brassicae' was shown to be the causal agent of blackleg on Chinese cabbage in China. We believe that this disease has historically often been misidentified as 'anthracnose' by local famers. The threat from L. biglobosa to the production of Chinese cabbage has been assessed. This accurate identification of the causal pathogen is a critical first step towards the development of disease management strategies.

LAMP Detection and Identification of the Blackleg Pathogen Leptosphaeria biglobosa 'brassicae'.

Blackleg of oilseed rape is a damaging invasive disease caused by the species complex Leptosphaeria maculans (Lm)/L. biglobosa (Lb), which is composed of at least two and seven phylogenetic subclades, respectively. Generally, Lm is more virulent than Lb, but under certain conditions, Lb can cause a significant yield loss in oilseed rape. Lb 'brassicae' (Lbb) has been found to be the causal agent for blackleg of oilseed rape in China, whereas Lm and Lb 'canadensis' (Lbc) were frequently detected in imported seeds of oilseed rape, posing a risk of spread into China. To monitor the blackleg-pathogen populations, a diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using a 615-bp-long DNA sequence from Lbb that was derived from a randomly amplified polymorphic DNA assay. The LAMP was optimized for temperature and time, and tested for specificity and sensitivity using the DNA extracted from Lbb, Lbc, Lm, and 10 other fungi. The results showed that the optimal temperature and time were 65°C and 40 min, respectively. The LAMP primer set was specific to Lbb and highly sensitive as it detected the Lbb DNA as low as 132 fg per reaction. The LAMP assay was validated using the DNA extracted from mycelia and conidia of a well-characterized Lbb isolate, and its utility was evaluated using the DNA extracted from leaves, stems, pods, and seeds of oilseed rape. The LAMP assay developed herein will help for monitoring populations of the blackleg pathogens in China and in developing strategies for management of the blackleg disease.

Molecular characterization of a novel botoulivirus from the phytopathogenic fungus Sclerotinia minor.

In this study, the complete genomic sequence of a novel botoulivirus (Sclerotinia minor botoulivirus 1, SmBV1) from the phytopathogenic fungus Sclerotinia minor strain LC45 was determined. The genome of SmBV1 is 2,882 nucleotides in length and contains a single large open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis showed that SmBV1 clustered with the botoulivirus clade within the family Botourmiaviridae. This is the first report of a botoulivirus in S. minor.

Strain identification and metabolites isolation of Aspergillus capensis CanS-34A from Brassica napus.

An isolate (CanS-34A) of Aspergillus from a healthy plant of oilseed rape (Brassica napus) was identified based on morphological characterization and multi-locus phylogeny using the sequences of internal transcribed spacer (ITS)-5.8S rDNA region, BenA (for ß-tubulin), CaM (for calmodulin) and RPB2 (for RNA polymerase II). The results showed that CanS-34A belongs to Aspergillus capensis Hirooka et al. The antifungal metabolites produced by CanS-34A in potato dextrose broth (PDB) were extracted with chloroform. Three antifungal metabolites were isolated and purified from the chloroform extract of the PDB cultural filtrates of CanS-34A, and chemically identified as methyl dichloroasterrate, penicillither and rosellichalasin. They all showed antifungal activity against the plant pathogenic fungi Botrytis cinerea, Monilinia fructicola, Sclerotinia sclerotiorum and Sclerotinia trifoliorum with the EC50 values ranging from 2.46 to 65.00 µg/mL. To our knowledge, this is the first report about production of penicillither by Aspergillus and about the antifungal activity of methyl dichloroasterrate, penicillither and rosellichalasin against the four plant pathogenic fungi.

Contrast Between Orange- and Black-Colored Sclerotial Isolates of Botrytis cinerea: Melanogenesis and Ecological Fitness.

Botrytis cinerea usually produces grayish mycelia and conidia as well as black-colored sclerotia (BS) due to accumulation of melanin. An isolate (XN-1) of B. cinerea producing orange-colored sclerotia (OS) on agar media was obtained from an orange-colored apothecium of an uncultured soil fungus. Whether or not the OS B. cinerea occurs on plants and how they differ from the BS isolates in melanogensis and ecological fitness remained unknown. This study, for the first time, confirmed the presence of the OS B. cinerea in strawberry and tomato plants that were surveyed in Hubei Province of China. Only five OS isolates were obtained from a total of 2,031 isolates surveyed from the two crops. The OS isolate XN-1 was compared and contrasted with the BS isolate B05.10 in sclerotial melanogenesis and ecological fitness. Sclerotial melanogenesis was evident in B05.10 but was deficient in XN-1. The OS were more susceptible to the two mycoparasites Trichoderma koningiopsis and Clonostachys rosea than the BS. The percentage of viable sclerotia after the mycoparasitism study was significantly (P < 0.01) lower in OS (21%) than in BS (48%). This study also reaffirmed the importance of melanization for survival of B. cinerea sclerotia.

Molecular characterization of a novel endornavirus from the phytopathogenic fungus Botrytis cinerea.

The complete sequence of a novel endornavirus (Botrytis cinerea endornavirus 1, BcEV1) from the phytopathogenic fungus Botrytis cinerea strain HBtom-372 was determined. The BcEV1 coding strand is 11,557 nucleotides long, possessing an open reading frame (ORF) that codes for a polyprotein of 3,787 amino acid residues and lacks a site-specific nick. The polyprotein contains viral methyltransferase (MTR) domain, a cysteine-rich region (CRR), two putative viral helicase (DEXDc-like and Hel-1) domains, and an RNA-dependent RNA polymerase_2 (RdRp_2) domain. In phylogenetic analysis, BcEV1 clustered with several fungal endornaviruses, forming an independent clade, and it was detected in 4.2 % of B. cinerea strains collected from central China.

Characterization of the Mycelial Compatibility Groups and Mating Type Alleles in Populations of Sclerotinia minor in Central China.

Ninety-five single-sclerotium isolates were obtained from lettuce and weeds in three counties in central China. They were identified belonging to Sclerotinia minor based on colony morphology and the S. minor-specific DNA marker. Mycelial compatibility groups (MCGs) and the mating type (MAT) alleles in these isolates were determined using the methods of paired cultures and specific PCR, respectively, and the MCG data were used to calculate Shannon's H index (H) and Simpson index (S), thereby evaluating diversity of S. minor. Eight MCGs (MCG1 to MCG8) and two MAT alleles (Inv+, Inv-) were identified in these isolates. Low diversity was detected for the total 95 isolates (H = 1.748, S = 0.786). Isolates of different MCGs or with different MAT alleles did not significantly differ (P > 0.05) in mycelial growth rate on potato dextrose agar (PDA, 20°C) or lesion diameter on lettuce leaves (20°C), but slightly differed in the number of sclerotia produced on PDA (20°C). Furthermore, this study reported five new host plants of S. minor in China, including Capsella bursa-pastoris, Oenanthe javanica, Fragaria gracilis, Ranunculus ternatus, and Salvia plebeia, and identified three hypovirulent isolates. These results broaden our understanding about the population biology of S. minor.

CmpacC regulates mycoparasitism, oxalate degradation and antifungal activity in the mycoparasitic fungus Coniothyrium minitans.

The PacC/Rim101 pH-responsive transcription factor is an important pathogenicity element for many plant-pathogenic fungi. In this study, we investigated the roles of a PacC homologue, CmpacC, in the mycoparasitic fungus Coniothyrium minitans. CmpacC was confirmed to have the transcriptional activation activity by the transcriptional activation test in Saccharomyces cerevisiae. Disruption of CmpacC resulted in impaired fungal responses to ambient pH. Compared to the wild-type, the CmpacC-disruption mutant ΔCmpacC-29 was significantly suppressed for activities of chitinase and ß-1,3-glucanase at pH 5 and 7, consistent with reduced expression levels of Cmch1 and Cmg1 coding for the two enzymes respectively. However, the mutant displayed acidity-mimicking phenotypes such as improved oxalate degradation and increased antifungal activity at pH 6 or higher. Improved efficacy in oxalate degradation by ΔCmpacC-29 was consistent with the enhanced expression level of Cmoxdc1 coding for oxalate decarboxylase. CmpacC transcriptional activation of Cmch1 and Cmg1 and repression of Cmoxdc1 were verified by the presence of the PacC/Rim101 consensus binding-motifs in gene promoter regions and by the promoter DNA-binding assays. This study suggests that CmpacC plays an activator role in regulation of C. minitans mycoparasitism, whereas plays a repressor role in regulation of oxalate degradation and possibly antifungal activity of C. minitans.

Novel hypovirulence-associated RNA mycovirus in the plant-pathogenic fungus Botrytis cinerea: molecular and biological characterization.

Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A -1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.

Effect of Water Flooding on Survival of Leptosphaeria biglobosa 'brassicae' in Stubble of Oilseed Rape (Brassica napus) in Central China.

Blackleg (Phoma stem canker) caused by Leptosphaeria maculans and L. biglobosa is an economically important disease on oilseed rape and many cruciferous vegetables. Oilseed rape-rice rotation is a routine cultivation practice in central China. This study was conducted to assess the effect of flooding on survival of L. biglobosa 'brassicae' in the stubble of winter oilseed rape (Brassica napus). Basal stems with typical blackleg symptoms were collected and cut into small pieces (2 cm) that were either submerged in water at 16 and 20, 20 and 28, 28 and 33, and 33 and 40°C (12 and 12 h) or kept dry at room temperature (control). Moreover, in a field experiment, the stem pieces were placed on the soil surface in a rice field or in a cotton field and either flooded in water or not flooded, respectively. After 1, 2, 4, 6, and 8 weeks, the stem pieces were sampled for retrieval of L. biglobosa 'brassicae' on V8-juice agar and for determination of dry weight. Selected L. biglobosa 'brassicae' isolates from the stem pieces were identified by polymerase chain reaction (PCR). Results from the two experiments showed that, compared with the controls, flooding for 1 to 2 weeks substantially reduced recovery of L. biglobosa 'brassicae' and flooding for 4 weeks resulted in negligible recovery of L. biglobosa 'brassicae'. All of the 99 selected isolates produced a 444-bp DNA fragment in the PCR, confirming that they belong to L. biglobosa 'brassicae'. Results also indicated that flooding caused rapid decomposition of the stem pieces. After flooding for 8 weeks, the dry weight of the stem pieces was reduced by 28 to 42% in the laboratory experiment and by 26 to 36% in the field experiment. These results suggest that oilseed rape-rice rotation is probably an efficient way to reduce longevity of L. biglobosa 'brassicae' in stubble of winter oilseed rape in central China.

Development of PCR-Based Assays for Detecting and Differentiating Three Species of Botrytis Infecting Broad Bean.

Botrytis cinerea, B. fabae, and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop polymerase chain reaction (PCR)-based assays to detect and differentiate these three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on two sequence-characterized amplified region markers derived from two random amplified polymorphic DNA assays. The other primer set, Bfa-f/Bfa-r for B. fabae, was designed based on the necrosis and ethylene-inducing protein 1 gene sequence. The three primer sets were highly specific for the corresponding species of Botrytis in both single and multiplex PCR assays. The PCR detection limit was 40, 40, and 400 pg of DNA per 25-µl reaction mixture for B. fabae, B. fabiopsis, and B. cinerea, respectively. Presence of the broad bean DNA in the PCR reactions at 1:1000 (Botrytis DNA/broad bean DNA [wt/wt]) had negligible effects on detection of the targeted Botrytis spp. The multiplex PCR assay was able to detect three Botrytis spp. in artificially infected and naturally infected broad bean leaves. These results suggest that the multiplex PCR assay developed in this study could be used to monitor the epidemics of chocolate spot of broad bean in the field.

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum.

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy-1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy-1, the Cmoxdc1-disrupted mutants had reduced expression levels of two mycoparasitism-related genes chitinase (Cmch1) and ß-1,3-glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1-disrupted mutants in OA-amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1-mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.

Characterization of a novel bipartite double-stranded RNA mycovirus conferring hypovirulence in the phytopathogenic fungus Botrytis porri.

The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. We report here the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 (5,879 bp), which share sequence identities of 62 and 95% at the 3'- and 5'-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were detected. The protein encoded by the 3'-proximal coding region of ORF I shows sequence identities of 19 to 23% with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae, Chrysoviridae, and Megabirnaviridae. However, the proteins encoded by the 5'-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of ~35 nm in diameter encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80, and 85 kDa, respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite dsRNA virus that possibly belongs to a new virus family.