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Pomegranate crown rot caused by Coniella granati is one of the most severe diseases of pomegranate. No fungicides have been registered for controlling this disease in China. Pyraclostrobin, belonging to strobilurin fungicides, has a broad spectrum of activity against many phytopathogens. In this study, based on the mycelial growth and conidial germination inhibition methods, we investigated the biological activity of pyraclostrobin against C. granati in the presence of 50 μg/ml of salicylhydroxamic acid using 80 isolates collected from different orchards in China from 2012 to 2018. The EC 50 (50% effective concentration) values ranged from 0.040 to 0.613 μg/ml for mycelial growth and 0.013 to 0.110 μg/ml for conidium germination. Treated with pyraclostrobin, the hyphae morphology changed and conidial production of C. granati decreased significantly. The result of transmission electron microscope showed that treatment of pyraclostrobin could make the cell wall thinner and lead to ruptured cell membrane and formation of intracellular organelle autophagosomes. The pyraclostrobin showed good protective and curative activities against C. granati on detached pomegranate fruits. In field trials, pyraclostrobin showed excellent control efficacy against this disease, in which the treatment of 25% pyraclostrobin EC 1,000× provided 92.25 and 92.58% control efficacy in 2019 and 2020, respectively, significantly higher than that of other treatments. Therefore, pyraclostrobin could be a candidate fungicide for the control of pomegranate crown rot.

New fungicides are tools to manage fungal diseases and overcome emerging resistance in fungal pathogens. In this study, a total of 121 Fusarium fujikuroi isolates were collected from various geographical regions of China and their sensitivity to a novel succinate dehydrogenase inhibitor (SDHI) fungicide 'pydiflumetofen' was evaluated. The 50% effective concentration (EC 50 ) value of pydiflumetofen for mycelial growth suppression ranged from 0.0101 to 0.1012 μg/ml and for conidial germination inhibition ranged from 0.0051 to 0.1082 μg/ml. Pydiflumetofen-treated hyphae showed contortion and increased branching, cell membrane permeability, and glycerol content significantly. The result of electron microscope transmission indicated that pydiflumetofen damaged the mycelial cell wall and the cell membrane, and almost broke up the cells, which increased the intracellular plasma leakage. There was no cross-resistance between pydiflumetofen and the widely used fungicides such as carbendazim, prochloraz, and phenamacril. Pydiflumetofen was found safe to seeds and rice seedlings of four rice cultivars, used up to 400 μg/ml. Seed treatment significantly decreased the rate of diseased plants in the greenhouse as well as in field trials in 2017 and 2018. Pydiflumetofen showed superb results against the rice bakanae disease (RBD), when used at 10 or 20 g a.i./100 kg of treated seeds, providing over 90% control efficacy (the maximum control efficacy was up to 97%), which was significantly higher than that of 25% phenamacril (SC) at 10 g or carbendazim at 100 g. Pydiflumetofen is highly effective against F. fujikuroi growth and sporulation as well as RBD in the field.

Lavender (Lavandula angustifolia Mill) is an ornamental plant and worldwidely grown for its aromatic and pharmacological qualities. In June 2020, the symptoms of blackleg disease on lavender plants were observed, with more than 50% incidence in Chaohu city (117°38'19.12″N, 31°47'18.94″W) of Anhui Province, China. The disease symptoms progressed from stem wilt and necrosis to prolonged necrosis and bending of leaves, and all infected lavender plants died eventually. Ten necrotic stem lesions werecollectedfrom ten independent plants for the isolation of pathogen. All samples were washed in 70% ethanol for 1 minute, rinsed twice in sterile distilled water and placed on water agar (WA) plates containing 30 mg/liter of kanamycin. All 16 fungal isolates were transferred onto potato dextrose agar (PDA) and incubated at 26°C for 5 days, and all fungal colonies were isolated consistently, which produced redish-gray mycelium at 26°C with a 12-h photoperiod on PDA media. They developed black pycnidia with abundant hyaline, unicellular, oval shaped conidia (4.5 to 5.9 × 2.1 to 2.5 μm) after 14 days. DNA was extracted (10-day-old culture) using the Fungal DNA Mini Kit (Omega Bio-tek, China), according to the manufacturer's protocol. The internal transcribed spacer (ITS), beta-tubulin (β-tub) and translation elongation factor 1-alpha (tef1-α) genes of three isolates were amplified using the primers: ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995) and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The ITS(MT883331), β-tub(MT896891) andtef1-α (MT874165) genes were sequenced and analyzed through BLASTn. The ITS sequence showed 99.81% with Epicoccum sorghinum (GenBank Accession No. MK020690.1). The β-tub and tef1-α showed 100% homology with Epicoccum sorghinum (GenBank AccessionMN554062.1 and MN512426.1), respectively. To complete Koch's postulates, pathogenicity tests were performed by spraying the fungal spore suspension (1×105 CFU/ml) prepared from 14-day-old cultures onto needling wounded stems of 1-year-old potted healthy L. angustifolia plants. The healthy plants were sprayed with sterilized water onto needling wounded stems served as negative control. Wilting and stem necrosis were observed 5 days afterinoculation and incubation in a growth chamber at 26°C, with a 12-h photoperiod. All fungal infected plants died after 10 days, while, the control plants remained healthy. The fungus was re-isolated from the lesions of the inoculated plants and verified. Based on morphological characteristics, sequence analysis and pathogenicity test, the pathogen was identified as E. sorghinum. The pathogen has been observed previously on many plants such as tea (Bao et al. 2019) and taro (Liu et al. 2018), in China. To our knowledge, this is the first report of E. sorghinum causing blackleg disease of lavender in China and worldwide.

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b ( cyt-b ) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl 2 (4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri , A. solani , and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.

Ustilaginoidea virens is an important fungus that causes rice false smut disease. This disease significantly reduces both grain yield and quality. Various methods have been developed for the detection of U. virens but most of these methods need sophisticated equipment such as a thermal cycler. Here, we present a loop-mediated isothermal amplification (LAMP) assay for the specific detection of U. virens. This assay used a specific region of the UvG-β1 gene (212-bp region) to design six LAMP primers. The LAMP assay was optimized by the combination of rapidity, simplicity, and high sensitivity for the detection of about 1 pg of target genomic DNA in the reaction whereas, with polymerase chain reaction (PCR), there was no amplification of DNA with concentrations less than 1 ng. Among the genomic DNA of 22 fungus species and two strains of U. virens, only the tube containing the DNA of U. virens changed to yellowish green with SYBR Green I. The color change was indicative of DNA amplification. No DNA was amplified from either the other 22 fungus species or the negative control. Moreover, 20 spikelets and 22 rice seed samples were used for the detection of rice false smut via LAMP. The results were comparable with conventional PCR. We conclude that gene UvG-β1 coupled with LAMP assay, can be used for the detection and identification of U. virens gene via LAMP.

Fusarium asiaticum is a causal agent of Fusarium head blight (FHB) of wheat in the southern part of China. Carbendazim has been extensively used for controlling FHB for more than 30 years, leading to the widespread carbendazim-resistant isolates in all major wheat-producing provinces in China, especially in Anhui Province. F. asiaticum isolates were collected throughout Anhui Province between 2010 and 2012 to monitor their sensitivity to carbendazim. In total, 74 of 899 single-spore isolates F. asiaticum were found to be resistant to carbendazim. Resistant isolates were collected from all of the sampled sites except Hefei of Anhui Province. The overall frequency of carbendazim resistance was shown to be 8.2%. Of the 74 isolates, 1, 68, and 5 had low resistance (LR), moderate resistance (MR) ,and high resistance (HR), respectively, to carbendazim. Five types of point mutations (F167Y, E198L, E198K, F200Y, and E198Q) in the β 2 -tubulin gene conferring resistance to carbendazim were detected in the field-resistant isolates with frequencies of 89.2, 2.7, 4.1, 2.7, and 1.4%, respectively. The point mutations at codon 167, 198, or 200 of the β 2 -tubulin gene were correlated with different levels of carbendazim resistance. Some of the sensitive and resistant isolates appeared to possess different biological characteristics; however, these might not be due to resistance. Because carbendazim resistance was generally widespread throughout Anhui Province, the sensitivity of F. asiaticum populations to carbendazim should be constantly monitored for the development of carbendazim resistance in natural populations.