Guoqing Li

Anilinopyrimidine (AP) fungicides have been widely adopted to control Botrytis cinerea since the 1990s. As a high-risk pathogen for the development of fungicide resistance, B. cinerea developed resistance to AP fungicides soon after their application. To ensure the proper use of these fungicides, it is necessary to establish a rapid and simple method for resistance detection. Our previous study demonstrated that the E407K mutation in Bcmdl1 was the major mutation conferring AP resistance in China. Based on the combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a nucleic acid detection assay (RPA/Cas12a detection assay), a simple method for the rapid detection of AP resistance was established by specifically identifying this resistance-related mutation. The new detection assay could precisely identify the E407K mutants from other mutants and wild-type isolates within 50 min, relying solely on a water/metal bath and a UV flashlight. Moreover, this assay could detect genomic DNA at a concentration as low as 1.8 × 10⁶ fg/μl, which is comparable with conventional PCR, indicating its high sensitivity. High specificity among different species was also observed in this assay. Above all, this assay was compatible with a 2-min DNA extraction method, implying its feasibility for field application. In conclusion, the RPA/Cas12a detection assay developed in this study is rapid and simple, making it an ideal method for AP resistance detection in local agencies and other points of care. Instant information on resistance monitoring can provide important guidance on resistance management.

Plenodomus biglobosus (Pb), a causal agent of blackleg of rapeseed, is composed of several subspecies, including ‘australensis’ (Pba), ‘brassicae’ (Pbb) and ‘canadensis’ (Pbc). Besides rapeseed, Pb can infect many wild cruciferous plants (WCPs), such as flixweed (Descurainia sophia) and pennycress (Thlaspi arvense), which may become the infection source for blackleg of rapeseed. However, Pb on WCPs has not been well investigated in China. This study identified the blackleg fungi on two WCPs in Sayram Lake and Zhaosu County in Xinjiang, China: flixweed (15 isolates) and pennycress (1 isolate) as well as on rapeseed (971 isolates). They belonged to Pba (11), Pbb (18), and Pbc (958). Pba occurred on flixweed (10) and pennycress (1) only in Sayram Lake, whereas Pbb and Pbc occurred on flixweed (1 and 4 isolates, respectively) and rapeseed (17 and 954 isolates, respectively) in Zhaosu County. Then, virulence of 16 isolates from flixweed and pennycress was determined on rapeseed. Their genomes were sequenced and used to identify the mating-type idiomorphs and to analyze population genetic structure. Results showed that all of the 16 isolates were virulent to rapeseed. Only MAT1-1 was detected in 11 Pba isolates, implying that Pba may lack sexual reproduction. The 16 isolates from two WCPs were divided into four genetic groups: Group I for Pbc (4 isolates), Group II for Pbb (1 isolate), and Group III (3 isolates) and IV (8 isolates) for Pba. The findings about the single mating type in Pba and its limited geographic distribution provided a case showing the importance of sexual reproduction in epidemics of Pb. To the best of our knowledge, this is the first report of Pba, Pbb, and Pbc on flixweed and Pba on pennycress in China.

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 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 the efficacy of the antimicrobials and P2 pycnidiospores in suppression of rapeseed clubroot. The results showed that cultural filtrates (CFs) 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 pycnidiospore suspension (P2 SS, 1 × 10⁷ spores/ml) reduced clubroot severity by 31 to 70% on the 30-day-old seedlings compared with the control (seeds treated with water). P2 was reisolated 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 with 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.

The fungal Gβ-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 Gβ-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gβ-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.

Eight fungal strains (P1 to P8) were isolated from rapeseed stems (Brassica napus) infected with the blackleg pathogen Leptosphaeria biglobosa (Lb). They formed pycnidia with similar morphology to those of Lb, and thus were considered as Lb relatives (LbRs). The species-level identification of these strains was performed. Their virulence on rapeseed and efficacy in the suppression of Lb infection were determined, and the biocontrol potential and biocontrol mechanisms of strain P2 were investigated. The results showed that the LbRs belong to two teleomorphic genera in the family Didymellaceae, Didymella for P1 to P7 and Boeremia for P8. Pathogenicity tests on rapeseed cotyledons and stems indicated the LbRs were weakly virulent compared to L. biglobosa. Co-inoculation assays on rapeseed cotyledons demonstrated that P1 to P7 (especially P1 to P4) had a suppressive effect on Lb infection, whereas P8 had a marginal effect on infection by L. biglobosa. Moreover, D. macrostoma P2 displayed a more aggressive behavior than L. biglobosa in the endophytic colonization of healthy rapeseed cotyledons. Cultures of P2 in potato dextrose broth (PDB) and pycnidiospore mucilages exuded from P2 pycnidia showed antifungal activity to L. biglobosa. Further leaf assays revealed that antifungal metabolites (AM) of strain P2 from PDB cultures effectively suppressed infection by L. biglobosa, Botrytis cinerea (gray mold), and Sclerotinia sclerotiorum (white mold). An antifungal metabolite, namely penicillither, was purified and identified from PDB cultures and detected in pycnidiospore mucilages of strain P2. This study suggests that the LbRs are a repertoire for screening biocontrol agents (BCAs) against rapeseed diseases, and D. macrostoma P2 is a multi-functional BCA, a penicillither producer, and an endophyte.

Phoma stem canker (blackleg), caused by the fungi Leptosphaeria maculans (anamorph Phoma lingam) and L. biglobosa, is one of the most devastating diseases in oilseed rape (Brassica napus L.) production worldwide. However, the population structure and genetic variation of L. biglobosa populations in China have rarely been investigated. Here, a collection of 214 fungal strains of blackleg from China (including Shaanxi, Jiangxi, Hubei, Jiangsu, Chongqing, Sichuan, Guangxi, Guizhou, Hunan, and Henan) and Europe (France and Ukraine) was identified as L. biglobosa. Three simple sequence repeat (SSR) markers were developed to characterize their population structure. The results showed that the Nei’s average gene diversity ranged from 0.6771 for the population from Jiangsu to 0.3009 for the population from Hunan. In addition, most of the genetic variability (96%) occurred within groups and there were only relatively small amounts among groups (4.0%) (F_ST = 0.043, p = 0.042 < 0.05). Pairwise population differentiation (F_ST) suggested that significant genetic differentiation was observed between different L. biglobosa populations. Bayesian and unweighted average method analysis revealed that these L. biglobosa strains were clustered into three branches, and three European strains were similar to those from eastern China. The pathogenicity assay showed that those in Group III were significantly more virulent than those in Group I (t = 2.69, p = 0.016). The study also showed that Group III was dominant in Chinese L. biglobosa populations, which provides new insights for the further study of population evolution and the management of this pathogen.

Sclerotinia sclerotiorum is an important plant pathogenic fungus of many crops. Our previous study identified the S. sclerotiorum agglutinin (SSA) that can be partially degraded by the serine protease CmSp1 from the mycoparasite Coniothyrium minitans. However, the biological functions of SSA in the pathogenicity of S. sclerotiorum and in its response to infection by C. minitans, as well as to environmental stresses, remain unknown. In this study, SSA disruption and complementary mutants were generated for characterization of its biological functions. Both the wild-type (WT) of S. sclerotiorum and the mutants were compared for growth and sclerotial formation on potato dextrose agar (PDA) and autoclaved carrot slices (ACS), for pathogenicity on oilseed rape, as well as for susceptibility to chemical stresses (NaCl, KCl, CaCl₂, sorbitol, mannitol, sucrose, sodium dodecyl sulfate, H₂O₂) and to the mycoparasitism of C. minitans. The disruption mutants (ΔSSA-175, ΔSSA-178, ΔSSA-225) did not differ from the WT and the complementary mutant ΔSSA-178C in mycelial growth. However, compared to the WT and ΔSSA-178C, the disruption mutants formed immature sclerotia on PDA, and produced less but larger sclerotia on ACS; they became less sensitive to the eight investigated chemical stresses, but more aggressive in infecting leaves of oilseed rape, and more susceptible to mycoparasitism by C. minitans. These results suggest that SSA positively regulates sclerotial development and resistance to C. minitans mycoparasitism, but negatively regulates pathogenicity and resistance to chemical stresses.

Damping-off caused by Pythium aphanidermatum (Pa) is one of the most destructive diseases for watermelon seedlings. Application of biological control agents against Pa has attracted the attention of many researchers for a long time. In this study, the actinomycetous isolate JKTJ-3 with strong and broad-spectrum antifungal activity was screened from 23 bacterial isolates. Based on the morphological, cultural, physiological, and biochemical characteristics as well as the feature of 16S rDNA sequence, isolate JKTJ-3 was identified as Streptomyces murinus. We investigated the biocontrol efficacy of isolate JKTJ-3 and its metabolites. The results revealed that seed and substrate treatments with JKTJ-3 cultures showed a significant inhibitory effect on watermelon damping-off disease. Seed treatment with the JKTJ-3 cultural filtrates (CF) displayed higher control efficacy compared to the fermentation cultures (FC). Treatment of the seeding substrate with the wheat grain cultures (WGC) of JKTJ-3 exhibited better control efficacy than that of the seeding substrate with the JKTJ-3 CF. Moreover, the JKTJ-3 WGC showed the preventive effect on suppression of the disease, and the efficacy increased with increase in the inoculation interval between the WGC and Pa. Production of the antifungal metabolite actinomycin D by isolate JKTJ-3 and cell-wall-degrading enzymes such as β-1,3-glucanase and chitosanase were probably the mechanisms for effective control of watermelon damping-off. It was shown for the first time that S. murinus can produce anti-oomycete substances including chitinase and actinomycin D. This is the first report about S. murinus used as biocontrol agent against watermelon damping-off caused by Pa.

Blackleg of oilseed rape caused by Leptosphaeria maculans/L. biglobosa is a worldwide important disease. L. maculans is more virulent than L. biglobosa, so it causes a great concern for oilseed rape production. In China, blackleg (L. biglobosa) of oilseed rape was reported in the 2000s, but epidemiological features of blackleg have not been well elucidated. Moreover, whether L. maculans exists in China is still an open question. Therefore, a 5-year survey was done in China to collect blackleg-occurrence data for characterizing the features of blackleg epidemics and to identify the blackleg pathogens for assessing the risk of L. maculans invasion. The results showed that all the 19 surveyed provinces had blackleg on oilseed rape, and the most frequently occurring provinces are Gansu, Qinghai, Shaanxi, and Hubei. Phoma stem canker was the most common symptom, which was associated with stem cracks on winter oilseed rape and with stem-weevil activities on spring oilseed rape. Temperature and rainfall were the main factors for blackleg epidemics on winter oilseed rape, whereas rainfall was the main factor for blackleg epidemics on spring oilseed rape. Brassica campestris and B. juncea oilseed rapes were more susceptible than B. napus to blackleg. Oilseed rapes cultivated under the continuous dry land-cropping pattern were more prone to blackleg than those cultivated under the paddy land/dry land-cropping pattern. All 6,015 fungal isolates from blackleg plant tissues belonged to L. biglobosa. These results are helpful for understanding the blackleg epidemics of oilseed rapes and for management of this disease in China.

Damping-off caused by Pythium aphanidermatum (Pa) is one of the most destructive diseases for watemelon seedlings. Application of biological control agents against Pa has attracted attention of many researchers for a long time. In this study, an actinomycete isolate JKTJ-3 with strong and broad-spectrum antifungal effect was screened from 23 bacterial isolates. Based on the morphological, cultural, physiological, and biochemical characteristics as well as the feature of 16S rDNA sequence, isolate JKTJ-3 was identified as Streptomyces murinus. We compared the biocontrol efficacy of isolate JKTJ-3 and its metabolites. The results revealed that seed and substrate treatments with the JKTJ-3 cultures showed a significant inhibitory effect on watermelon damping-off disease. Seed treatment with the JKTJ-3 cultural filtrates (CF) had higher control efficacy that of the fermentation cultures (FC). Treatment of the seeding substrate with the wheat grain cultures (WGC) of JKTJ-3 exhibited better control efficacy than that of the seeding substrate with the JKTJ-3 CF. Moreover, the JKTJ-3 WGC showed the preventive effect on suppression of the disease, and the efficacy increased with increase in the inoculation interval between the WGC and Pa. Production of the antifungal metabolite actinomycin D by isolate JKTJ-3 and cell wall-degrading enzymmes such as β-1,3-glucanase and chitosanase was probably the mechanisms for effective control of watermelon damping-off. It was showed for the first time that S. murinus can produce anti-oomycete substances including chitinase and actinomycin D. This is the first report about S. murinus used as biocontrol agent against watermelon Pa-induced damping-off.