Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Cu/Zn superoxide dismutase (VdSOD1) mediates reactive oxygen species detoxification and modulates virulence in Verticillium dahliae.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn-SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione-treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more O2- and less H2 O2 than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host-generated extracellular ROS, and contributes significantly to virulence in V. dahliae.

The Whole Genome Sequence of Fusarium redolens Strain YP04, a Pathogen that Causes Root Rot of American Ginseng.

Fusarium redolens was previously reported as a plant pathogen or an endophyte that is closely related to F. oxysporum, a notoriously significant soilborne phytopathogen. Subsequent studies demonstrated the unique nature of F. redolens, which was considered a distinct species that causes multiple symptoms on multiple hosts. It was recently identified as a pathogen that causes root rot of American ginseng. Currently, few high-quality F. redolens genome sequences exist in the public database. Here, we report the whole-genome sequence of F. redolens strain YP04, based on a hybrid assembly of long- and short-read sequencing with PacBio and Illumina platforms, respectively. The assembly consists of 40 configs with a total length of 52.8 Mb nuclear genomic DNA and 49.6 kb complete mitochondrial genomic DNA, and encodes a total of 18,985 genes, including 18,517 protein-coding genes and 469 RNA genes which were functionally annotated. In total, 4,606 proteins were identified in the pathogen-host interactions database, suggesting that they were likely involved in pathogenicity and host-pathogen interactions, while 41 secondary metabolite synthesis clusters were predicted and annotated. This is the first high-quality whole genome of F. redolens, providing an important community resource for genome evolution, host-pathogen interaction, and secondary metabolite biosynthesis studies.

Assessment of Resistance in Potato Cultivars to Verticillium Wilt Caused by Verticillium dahliae and Verticillium nonalfalfae.

Verticillium wilt caused by Verticillium spp., also called potato early dying disease, is one of the most serious soilborne diseases affecting potato production in China. The disease has been expanding into most potato production areas over the past few years. Information on host resistance against Verticillium wilt among the potato cultivars in China is scarce, but it is critical for sustainable management of the disease. This study, therefore, evaluated 30 commercially popular potato cultivars against Verticillium dahliae strain Vdp83 and Verticillium nonalfalfae strain Vnp24, two well-characterized strains causing Verticillium wilt of potato in China. Both strains were isolated from diseased potato plants, and they were previously proven to be highly virulent. Ten plants of each cultivar were inoculated with the V. dahliae strain and incubated on greenhouse benches. Symptoms were rated at weekly intervals, and the relative area under the disease progress curve was calculated. The experiment was repeated once, and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals. Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V. nonalfalfae strain. Cultivar responses to V. nonalfalfae were like those exhibited against V. dahliae, except for one cultivar. This study showed that resistance among potato cultivars exists in China against Verticillium spp. and that the resistance to V. dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions. Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study.

Genetic Diversity of Verticillium dahliae Populations From Olive and Potato in Lebanon.

Verticillium dahliae is widely distributed in potato and olive fields in Lebanon, causing serious economic losses. However, little is known about the inoculum source, population structure, and genetic diversity of the pathogen or the mechanisms of dissemination within Lebanon. To understand the population structure, a total of 203 isolates sampled from olive (n = 78) and potato (n = 125) were characterized for species, mating type, and race, and the genetic relationships were delineated using 13 microsatellite markers. All isolates except one from potato were V. dahliae, with 55.1 and 12.1% race 1, and 43.6 and 83.1% race 2 in olive and potato, respectively. The genetic structure of the studied population was best described by two large and two small clusters. Membership in the two large clusters was determined by the presence or absence of the effector gene Ave1. Furthermore, genetic structure was moderately associated with the host of origin but was weakly associated with the geographic origin. All but four isolates represented by three multilocus haploid genotypes were MAT1-2. This study identified a clear lack of gene flow between virulence genotypes of V. dahliae despite the proximity of these cropping systems and the wide distribution of genetic diversity among hosts and geographic regions in Lebanon.

Verticillium Wilt Caused by Verticillium dahliae and V. nonalfalfae in Potato in Northern China.

Potato (Solanum tuberosum L.) is one of the most important staple foods in many parts of the world including China. In recent years, Verticillium wilt has become a severe threat to potato production in China. During 2015 to 2016, 287 samples of symptomatic potato plants were collected from 15 counties in five provinces from northern China. One hundred and eighty-seven Verticillium-like colonies were isolated from these samples and identified to species based on cultural and morphological characteristics, and multigene phylogeny based on the partial sequences of actin (ACT), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS) genes. A consensus-rooted most parsimonious phylogenetic tree was generated from the data. One hundred and fifteen isolates comprising 61.5% of the total were identified as Verticillium dahliae, and the remaining 38.5% of the isolates were identified as V. nonalfalfae. V. dahliae was widely distributed in Shaanxi (84.1%), Inner Mongolia (76.7%), Gansu (12.8%), and Qinghai (100%, representing a single isolate). V. dahliae was not recovered from the samples in Ningxia. V. nonalfalfae dominated the collections from Gansu (87.2%) and Ningxia (100%) but was also recovered from Shaanxi (15.9%) and Inner Mongolia (23.3%) at lower frequencies. Neither V. albo-atrum nor V. alfalfae was recovered from the sampled areas. The V. nonalfalfae isolates were predominantly isolated from the samples collected from altitudes above 1,800 m, and in contrast, V. dahliae isolates were mainly recovered from fields sampled below 1,800 m. The optimum temperature for the colony growth of V. nonalfalfae was lower (20°C) than that for V. dahliae (25°C). Pathogenicity tests demonstrated that V. dahliae and V. nonalfalfae were both pathogens of potato Verticillium wilt, with V. dahliae isolates exhibiting higher virulence than V. nonalfalfae isolates regardless of the collection area of the species. This is the first documentation of V. nonalfalfae infecting S. tuberosum in China and the higher altitudes associated with infections of V. nonalfalfae anywhere in the world.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii).

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

The heterothallic sugarbeet pathogen Cercospora beticola contains exon fragments of both MAT genes that are homogenized by concerted evolution.

Dothideomycetes is one of the most ecologically diverse and economically important classes of fungi. Sexual reproduction in this group is governed by mating type (MAT) genes at the MAT1 locus. Self-sterile (heterothallic) species contain one of two genes at MAT1 (MAT1-1-1 or MAT1-2-1) and only isolates of opposite mating type are sexually compatible. In contrast, self-fertile (homothallic) species contain both MAT genes at MAT1. Knowledge of the reproductive capacities of plant pathogens are of particular interest because recombining populations tend to be more difficult to manage in agricultural settings. In this study, we sequenced MAT1 in the heterothallic Dothideomycete fungus Cercospora beticola to gain insight into the reproductive capabilities of this important plant pathogen. In addition to the expected MAT gene at MAT1, each isolate contained fragments of both MAT1-1-1 and MAT1-2-1 at ostensibly random loci across the genome. When MAT fragments from each locus were manually assembled, they reconstituted MAT1-1-1 and MAT1-2-1 exons with high identity, suggesting a retroposition event occurred in a homothallic ancestor in which both MAT genes were fused. The genome sequences of related taxa revealed that MAT gene fragment pattern of Cercospora zeae-maydis was analogous to C. beticola. In contrast, the genome of more distantly related Mycosphaerella graminicola did not contain MAT fragments. Although fragments occurred in syntenic regions of the C. beticola and C. zeae-maydis genomes, each MAT fragment was more closely related to the intact MAT gene of the same species. Taken together, these data suggest MAT genes fragmented after divergence of M. graminicola from the remaining taxa, and concerted evolution functioned to homogenize MAT fragments and MAT genes in each species.