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A defence response can be induced by nonpathogenic Fusarium oxysporum CS-20 in several crops, but the molecular mechanism has not been clearly demonstrated. In the present study, we analysed the defence mechanism of a susceptible cucumber cultivar ( Cucumis sativus L. 9930) against a pathogen ( F. oxysporum f. sp. cucumerinum) through the root precolonization of CS-20. A challenge inoculation assay indicated that the disease severity index (DSI) was reduced, ranging from 18.83 to 61.67 in comparison with the pathogen control. Root colonization analysis indicated that CS-20 clearly did not appear to influence the growth of cucumber seedlings. Quantitative reverse-transcriptase polymerase chain reaction ( qRT-PCR) revealed that CS-20-mediated defence response was activated by PR3, LOX1 and PAL1 and the pathogen-mediated resistance response was regulated by PR1 and PR3. Moreover, both nonpathogenic and pathogenic F. oxysporum were able to upregulate NPR1 expression. In contrast to a pathogen, CS-20 can activate the Ca2+/CaM signal transduction pathway, and the gene expression of both CsCam7 and CsCam12 increased significantly. The gene expression analysis indicated that CS-20 strongly enhanced the expression of PR3, LOX1, PAL1, NPR1, CsCam7 and CsCam12 after inoculation. Overall, the defence response induced by CS-20 can be controlled by multiple genes in the cucumber plant. [ABSTRACT FROM AUTHOR]

Abstract: The Phytophthora capsici inflicts damage on numerous crop plants by secreting a series of pectinase including pectin methylesterase (PME). We identified a PME gene (pcpme6) from a genomic library of a highly virulent P. capsici strain SD33 which had an encoded a polypeptide of 348 amino acid residues with a predicted molecular mass of 38.18 kDa. We also confirmed that pcpme6 was increasingly expressed during symptom development following P. capsici infection of pepper leaves. The wild-type protein (PCPME6) ca. 50 kDa was obtained from pcpme6 expression, and PME activity trend in PCPME6-treated pepper leaves increased with symptom development. PCPME6 degraded leaf cell walls, resulting in the production of necrotic lesions. Mutation of Asp residues in active sites within pcpme6 affected PCPME6 activity and its virulence on pepper leaves. Results show that pcpme6 is a gene within the pme gene family that is important for pathogenesis of P. capsici on pepper. [Copyright &y& Elsevier]

In recent years, 'Shine Muscat' ( Vitis labruscana × Vitis vinifera ) has been the most popular table grape among consumers in Asian countries (Choi et al., 2021). In November 2020, fruit rot was observed in packing house 2 months after cold-stored in Yuncheng (35°16'N, 111°08'E), Shanxi Province, China. Of 38 boxes (5 kg per box) grape were selected randomly from the packing house with a stock of 7,000 kg. The incidence of grape rot was 71.5%, observed in 143 of the 200 clusters sampled. The diseased fruit showed symptoms including pedicel browning, and extending lesions and softening of berries. Samples (1 mm2) cut from the border between diseased and healthy tissue were surface sterilized with 70% ethanol for 1 min, then rinsed with sterile distilled water thrice, and placed on potato dextrose agar (PDA) plate 25 ° C in the dark for 5 days. After subculturing for three times, 26 isolates with a similar type of fungal colony were obtained. Colonies on PDA was initially white fluffy aerial, but gradually turned grey. Pycnidia were black and globes, producing α and β conidia. α conidia abundant, aseptate, hyaline, ellipsoid, both ends obtuse, biguttulate, 6.52~9.05 μm×2.19~3.25 μm (n=50). β conidia abundant, hyaline, aseptate, filiform, flexuous to J-shaped, 22.79~36.75 μm×1.24~1.92 μm (n=50). For further identification, phylogenetic analysis of combined internal transcribed spacer (ITS), β-tubulin (TUB), translation elongation factor (EF1-α) and calmodulin (CAL) datasets were carried out. The representative isolate SS1 and SS2 were selected for identification. Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS region. The gene fragments of TUB, CAL and EF1-α were amplified by the primers Bt2a/Bt2b (Glass and Donaldson 1995), CAL228F/CAL737R and EF1-728F/EF1-986R, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank (SS1: MW644526 for ITS, OQ718912 - 4 for CAL, TEF-1α, and TUB, respectively; SS2: MW644527 for ITS, OQ718915 -7 for CAL, TEF-1α, and TUB, respectively). BLASTn analysis of the NCBI database indicated that the ITS, TUB, EF1-α, and CAL sequences had very high nucleotide homology (98 to 100%) to ex-type sequences for D. nobilis (CBS 116953, Gomes et al., 2013) in GenBank (ITS: KC343147, TUB: KC344115, EF1-α: KC343873, and CAL: KC343389). Phylogenetic tree based on the combined sequences revealed that the two isolates clustered well with D. nobilis. To confirm its pathogenicity, 30 fresh-harvested 'Shine Muscat' grape fruits were used to mock inoculate. Wounds (5 mm wide by 2 mm deep) were created uniformly at the equator of the grape fruit with a sterile puncher and inoculated with mycelial plugs (5 mm in diameter) prepared from 7-day-old fungal cultures grown on PDA (Wild 1994). The control fruits were inoculated with agar disks without mycelium. Inoculated fruits were placed in a chamber with the 80% relative humidity at 25°C for 8 days. After inoculation, all treated fruits turned brown, with significant necrotic lesions; fruits in control showed no symptoms. Fungal colonies reisolated from inoculated fruits had similar morphological characteristics as D. nobilis. Diaporthe spp. are responsible for diseases on a wide range of plants hosts, causing root and fruit rots, dieback, cankers, leaf spots, blights, decay and wilt (Gomes et al., 2013). D. nobilis causing postharvest grape rot can seriously restrict the development of local grape industry.

Gray mold caused by Botrytis cinerea is detrimental to plants and fruits. Endophytes have been shown to modify plant disease severity in functional assays. We conducted this study to investigate the endophytic strain Bacillus K1 with excellently antagonistic B. cinerea from the wild grape endosphere. We identified a wild grape endophytic strain K1 with high antifungal activity against B. cinerea both in vitro and in vivo . Combining the phylogenetic results based on 16S rDNA and genome sequencing, K1 was assigned as Bacillus subtilis . The in vitro results displayed that K1 and its volatile substances could significantly inhibit the mycelia growth of B. cinerea . Grape fruit inoculated with Bacillus K1 showed lower gray mold during treatment. The higher levels of defense-related enzymes, including peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase, were induced in grapes after inoculation. Scanning electron microscopy (SEM) suggested that K1 inhibited mycelial growth via bacterial colonization and antibiosis in grapes. The gas chromatography-mass spectrometry analysis identified 33 volatiles in which dibutyl phthalate was the major compound accounting for 74.28%. Dibutyl phthalate demonstrated strong activity in suppressing the mycelia growth of B. cinerea . Genome bioinformatics analysis revealed that the K1 chromosome harbored many known biosynthesis gene clusters encoding subtilosin, bacillaene, bacillibactin, bacilysin, and fengycin. This study provides a potential biological agent to control diseases of post-harvest grape fruit and improves our understanding of the possible biocontrol mechanisms of the Bacillus strain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Feng, Yao, Wei, Zhao and Shi.)

Velvet genes play critical roles in the regulation of diverse cellular processes. In current study, we identified the gene FocVel1, a homolog of Fusarium graminearum VelA, in the plant pathogenic fungus F. oxysporum f. sp. cucumerinum. This pathogen causes the destructive disease called cucumber Fusarium wilt (CFW), which severely affects the production and marketing of this vegetable worldwide. Transcript analyses revealed high expression of FocVel1 during conidiophore development. Disruption of the FocVel1 gene led to several phenotypic defects, including reduction in aerial hyphal formation and conidial production. The deletion mutant ΔFocVel1 showed increased resistance to both osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and prochloraz fungicides, which may be related to changes in cell wall components. In the process of biofilm formation in vitro, the mutant strain ΔFocVel1 displayed not only a reduction in spore aggregation but also a delay in conidial germination on the polystyrene surface, which may result in defects in biofilm formation. Moreover, pathogenicity assays showed that the mutant ΔFocVel1 exhibited impaired virulence in cucumber seedlings. And the genetic complementation of the mutant with the wild-type FocVel1 gene restored all the defects of the ΔFocVel1. Taken together, the results of this study indicated that FocVel1 played a critical role in the regulation of various cellular processes and pathogenicity in F. oxysporum f. sp. cucumerinum.

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 °C, respectively. The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.

Phytophthora capsici causes damage on many plants species, and secretes various pectin methylesterases during all stages of infection. We identified nine Pme genes (Pcpme 1-9) from a genomic library of highly virulent P. capsici strain SD33 and further analyzed the expression pattern of nine genes on three hosts: pepper, tomato, and cucumber using qRT-PCR during all stages of infection. All nine genes were found to be differentially expressed in three host species in the course of P. capsici interaction. The expression levels of the respective genes increased from 1 to 7 dpi in pepper, while most genes presented a decreasing trend of expression from 1 to 5 dpi in tomato fruits. However, in both fruits peaks were reached at 7 dpi. In cucumber fruits, each gene showed minor expression levels from 1 to 3 dpi, exhibited definite peaks at 5 dpi, and then decreased from 5 to 7 dpi. Thus, evidence from our studies of Pcpme gene expression in different plants at a rang of time points suggests that the late stages of infection may represent the most critical time for P. capsici to successfully express or/and secret PMEs., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)