Soil-borne Fungal Pathogens Research Articles

Detection of Mycovirus in Sclerotium Rolfsii, A Phytopathogenic Fungus in Bangladesh

A research project was conducted to detect presence of mycovirus, a probable virocontrol agent in Sclerotium rolfsii, a destructive soil-borne plant pathogenic fungus. A total of 500 isolates of the pathogen was isolated from infected wheat, bush bean, tomato, eggplant, lentil, chickpea, eggplant, chilli and okra grown Bangladesh following tissue planting methods and brought to the Virology laboratory of Okayama University, Japan. After brought to Japan, a total of 472 isolates were re-isolated and the fungal isolates were cultured on potato dextrose agar for extraction of DNA and dsRNA. The extracted nucleic acids were tested for the presence of viruses using rolling circle amplification (RCA) (for DNA viruses) and cellulose column chromatography (for RNA viruses). The detected S. rolfsii viruses were further characterized molecularly. For detection of DNA virus, a total of 120 isolates were tested, none were found positive for DNA viruses. For detection of dsRNA virus, a total of 472 isolates were tested, 7 isolates showed possible dsRNA positive band. The partial cDNA sequences of the dsRNA segments isolated from the strain SR336 were obtained. BlastX database search with the partial sequence from SR336 isolates showed similarities with randorna like virus. To screen the presence of randorna like virus in S. rolfsii isolates, 64 isolates were selected from 472 isolates of S. rolfsii. The isolates of S. rolfsii were tested by RT-PCR using randorna virus specific primers. Out of 64 isolates 13, (20.31%) isolates were randorna virus positive strain and rest of the isolates were absent of randorna virus.
 Bangladesh J. Agril. Res. 46(3): 331-341, September 2021

Dose-response of pests to ethanedinitrile dose-response of weed seeds, soil borne pathogens, and plant-parasitic nematodes to ethanedinitrile

Ethanedinitrile is a chemical soil fumigant with promising efficacy against several key pests including weeds, nematodes, and soil-borne pathogens. The efficacy of 12 concentrations of ethanedinitrile, ranging from 8.7 to 1,751 mg kg− 1 soil, to control seeds and tubers of five weed species (Cyperus esculentus, C. rotundus, Malva parviflora, Portulaca oleracea, and Stellaria media), two nematode species (Globodera rostochiennsis and Tylenchulus semipenetrans), and four pathogenic fungal species (Fusarium oxysporum, Macrophomina phaseolina, Pythium ultimum, and Verticillium dahlia) were evaluated in acidic sand (pH: 5.6) and alkaline sandy loam (pH: 7.5–7.6), under controlled laboratory conditions. These pathogens and weeds are common in strawberry and vegetable fields and have been targeted by soil fumigants. Ethanedinitrile was injected into microcosms for 24 h. Lower doses of ethanedinitrile controlled fungal pathogens and nematodes better in acidic sand than in alkaline sandy loam. However, the reverse tended to be true for weed control as higher doses of ethanedinitrile in the acidic sand were required to control weeds than in the alkaline sandy loam. Results showed that ethanedinitrile can provide excellent control of soil-borne nematodes, pathogenic fungi, and key weed species in acidic sand and alkaline sandy loam.

Introgression of the QTL qSB11-1TT conferring sheath blight resistance in rice (Oryza sativa) into an elite variety, UKMRC 2, and evaluation of its backcross-derived plants.

Sheath blight (SB) is the most damaging fungal disease in rice caused by a soil-borne pathogenic fungus, Rhizoctonia solani Kuhn (R. solani). The disease resistance in rice is a complex quantitative trait controlled by a few major genes. UKMRC2 is a newly developed elite rice variety that possesses high yield potential but is susceptible to sheath blight disease indicating a huge risk of varietal promotion, mass cultivation, and large-scale adoption. The aim of our present study was the development of varietal resistance against R. solani in UKMRC2 to enhance its stability and durability in a wide range of environments and to validate the effects of an SB-resistance QTL on the new genetic background. In our study, we developed 290 BC1F1 backcross progenies from a cross between UKMRC2 and Tetep to introgress the QTL qSBR11-1TT into the UKMRC2 genetic background. Validation of the introgressed QTL region was performed via QTL analysis based on QTL-linked SSR marker genotyping and phenotyping against R. solani artificial field inoculation techniques. The QTL qSBR11-1TT was then authenticated with the results of LOD score (3.25) derived from composite interval mapping, percent phenotypic variance explained (14.6%), and additive effect (1.1) of the QTLs. The QTL region was accurately defined by a pair of flanking markers K39512 and RM7443 with a peak marker RM27360. We found that the presence of combination of alleles, RM224, RM27360 and K39512 demonstrate an improved resistance against the disease rather than any of the single allele. Thus, the presence of the QTL qSBR11-1TT has been validated and confirmed in the URMRC2 genetic background which reveals an opportunity to use the QTL linked with these resistance alleles opens an avenue to resume sheath blight resistance breeding in the future with marker-assisted selection program to boost up resistance in rice varieties.

Heterosis in root microbiota inhibits growth of soil-borne fungal pathogens in hybrid rice.

In nature, plants are colonized by various microbes that play essential roles in their growth and health. Heterosis is a natural genetic phenomenon whereby first-generation hybrids exhibit superior phenotypic performance relative to their parents. It remains unclear whether this concept can be extended to the "hybridization" of microbiota from two parents in their descendants and what benefits the hybrid microbiota might convey. Here, we investigated the structure and function of the root microbiota from three hybrid rice varieties and their parents through amplicon sequencing analysis of bacterial 16S ribosomal DNA (rDNA) and fungal internal transcribed spacer (ITS) regions. We show that the bacterial and fungal root microbiota of the varieties are distinct from those of their parental lines and exhibit potential heterosis features in diversity and composition. Moreover, the root bacterial microbiota of hybrid variety LYP9 protects rice against soil-borne fungal pathogens. Systematic analysis of the protective capabilities of individual strains from a 30-member bacterial synthetic community derived from LYP9 roots indicated that community members have additive protective roles. Global transcription profiling analyses suggested that LYP9 root bacterial microbiota activate rice reactive oxygen species production and cell wall biogenesis, contributing to heterosis for protection. In addition, we demonstrate that the protection conferred by the LYP9 root microbiota is transferable to neighboring plants, potentially explaining the observed hybrid-mediated superior effects of mixed planting. Our findings suggest that some hybrids exhibit heterosis in their microbiota composition that promotes plant health, highlighting the potential for microbiota heterosis in breeding hybrid crops.

Virulence Spectrum among Rhizoctonia solani f. sp. sasakii Isolates from Multi-Geographical Locations of India

Rhizoctonia solani f. sp. sasakii (RSS) is a highly devastating soil-borne fungal pathogen inciting banded leaf and sheath blight (BLSB) disease in maize. In India, annually one percent of the total maize yield is reduced by BLSB. Due to continuous occurrence and wide spread of disease, the present experiment was conducted to assess the pathogenic variability among different isolates obtained from multi-geographical locations of India. Pathogenic behaviour of each isolates was predicted through the comparable study of their incubation period, average disease score and disease intensity on the three maize hybrids (Normal maize- HM 8, QPM maize- QPM 9 and sweet corn- HSC I). The incubation period of all the R. solani isolates ranged from 3 to 7 days with average disease score ranged from 2.1 to 7.1depending upon the host and isolate interaction after artificial inoculation. Disease intensity of all the RSS isolates on maize hybrids varied from 8.1 to 52.1% while isolates RSS 29 (New Delhi) and RSS 1 (Karnal) were found highly virulent, in comparison to other forty nine RSS isolates. As these isolates were highly virulent on maize hybrids with average disease score ranged from 6.2 to 7.1, whereas the isolates RS 30 (Dholi) and RS 31 (Dhaula Kuan) were recorded as least virulent, showed resistant disease reaction with average disease score ranged from 1.5 to 2.4.

Predatory protists play predominant roles in suppressing soil-borne fungal pathogens under organic fertilization regimes

Soil-borne fungal pathogens pose a major threat to global agricultural production and food security. Pathogen-suppressive bacteria and plant beneficial protists are important components of soil microbiomes and essential to plant health and performance, but it remains largely unknown regarding how agricultural management practices influence the relative importance of protists and bacteria in plant disease suppression. Here, we characterized soil microbiomes (including fungi, protists, and bacteria) in bulk and sorghum rhizosphere soils with various long-term inorganic and organic fertilization regimes, and linked the changes in fungal plant pathogens with the protistan and bacterial communities. We found that the relative abundances of fungal pathogens were significantly decreased by organic fertilization regimes, and there was a significant difference in the community composition of fungal pathogens between inorganic and organic fertilization regimes. Organic fertilization significantly enhanced predatory protists but reduced the proportions of protistan phototrophs. Co-occurrence network analysis revealed more intensive connections between fungal plant pathogens with protists, especially predatory protists, than with bacterial taxa, which was further supported by stronger associations between the community structure of fungal pathogens and predatory protists. We identified more protist consumer taxa than bacterial taxa as predictors of fungal plant pathogens, and structural equation modelling revealed a more important impact of protist consumers than bacteria on fungal pathogens. Altogether, we provide new evidence that the disease inhibitory effects of long-term organic fertilization regimes could be best explained by the potential predation pressure of protists. Our findings advance the mechanistic understanding of the role of predator-prey interactions in controlling fungal diseases, and have implications for novel biocontrol strategies to mitigate the consequences of fungal infections for plant performance.

A panel of qPCR assays to detect and quantify soybean soil-borne pathogens.

Fusarium oxysporum,F. graminearum,F. acuminatum,F. equiseti,F. proliferatum,F. solani, and Rhizoctonia solani are soil-borne fungal pathogens that cause substantial yield loss in a widespread list of crops worldwide. The objective of this study was to develop a panel of TaqMan assays for the detection and quantification of these six widespread soil-borne fungal species using real-time polymerase chain reaction (qPCR). The primers and probes were designed based on the intergenic spacer ribosomal RNA and translation elongation factor 1-alpha gene (tef1). These assays, although not multiplexed, can be performed simultaneously as they have similar reaction conditions, allowing more efficiency when targeting multiple pathogens in a sample. The assays presented high efficiency (94.3%-108.9%) and sensitivity, with a limit of detection of 0.05 picograms (50 femtograms) of target DNA. Results from an assay targeting 19 non-target and closely related species confirmed the specificity of the developed assays. The assays were also evaluated to detect the target species in different matrices, such as soil and plant material. This panel of qPCR assays is an additional tool that can be used by plant pathologists, microbiologists, plant breeders, diagnostic clinics, and other researchers interested in these fungal species.

Volatile organic compounds shape belowground plant-fungi interactions.

Volatile organic compounds (VOCs), a bouquet of chemical compounds released by all life forms, play essential roles in trophic interactions. VOCs can facilitate a large number of interactions with different organisms belowground. VOCs-regulated plant-plant or plant-insect interaction both below and aboveground has been reported extensively. Nevertheless, there is little information about the role of VOCs derived from soilborne pathogenic fungi and beneficial fungi, particularly mycorrhizae, in influencing plant performance. In this review, we show how plant VOCs regulate plant-soilborne pathogenic fungi and beneficial fungi (mycorrhizae) interactions. How fungal VOCs mediate plant-soilborne pathogenic and beneficial fungi interactions are presented and the most common methods to collect and analyze belowground volatiles are evaluated. Furthermore, we suggest a promising method for future research on belowground VOCs.

A highly polymorphic effector protein promotes fungal virulence through suppression of plant-associated Actinobacteria.

Plant pathogens secrete effector proteins to support host colonization through a wide range of molecular mechanisms, while plant immune systems evolved receptors to recognize effectors or their activities to mount immune responses to halt pathogens. Importantly, plants do not act as single organisms, but rather as holobionts that actively shape their microbiota as a determinant of health. The soil-borne fungal pathogen Verticillium dahliae was recently demonstrated to exploit the VdAve1 effector to manipulate the host microbiota to promote vascular wilt disease in the absence of the corresponding immune receptor Ve1. We identify a multiallelic V.dahliae gene displaying c. 65% sequence similarity to VdAve1, named VdAve1-like (VdAve1L), which shows extreme sequence variation, including alleles that encode dysfunctional proteins, indicative of selection pressure to overcome host recognition. We show that the orphan cell surface receptor Ve2, encoded at the Ve locus, does not recognize VdAve1L. Additionally, we demonstrate that the full-length variant VdAve1L2 possesses antimicrobial activity, like VdAve1, yet with a divergent activity spectrum, that is exploited by V.dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota. Our findings open up strategies for more targeted biocontrol against microbial plant pathogens.

Maize fungal root pathogens as affected by fertilisation and rotation with legumes

The abilities of grain legumes to fix atmospheric nitrogen are well documented, but the non-N effects of incorporating legumes into cereal-based systems are poorly understood. Continuous cropping of maize is assumed to increase maize fungal root pathogens, but few studies have documented this in sub-Saharan Africa. In this study, it was hypothesized that maize pathogen densities of F. oxysporum, F. verticillioides, F. equiseti, F. graminearum, F. chlamydosporum, C. eragostidis, M. phaseolina, Trichoderma spp., Pythium spp, Phoma spp, R. solani and E.pedicellatum are lower under legume-maize rotations than under continuous maize cropping, while fertilisation also affects pathogen densities. Field measurements were taken during the 2017/18 and 2018/19 summer growing seasons at two sites in South Africa to investigate the effect of crop rotation and fertilisation on densities of soil-borne fungal pathogens in maize. Densities of 12 pathogens on maize roots and crowns were investigated 110 days after planting using a modified DNA extraction method and qPCR technology. Maize following a legume had lower densities of F. graminearum, F. oxysporum, E. pedicellatum, and C. eragostidis at both localities. However, no consistent rotation effects were observed on densities of M. phaseolina, Trichoderma spp., Pythium spp., R. solani, Phoma spp., F. verticillioides and F. equiseti. Impacts of fertilisation on pathogen densities were also inconsistent depending on the site and season. This warrants for an integrated approach to fungal pathogen control in maize production.

Research Progress in Detection and Control of Soil Borne Disease Fusarium Root Rot

Fusarium is a variety of species, which is difficult to distinguish in morphology. Traditionally, identification of pathogenic fusarium requires the separation of pathogenic bacteria, purification of pathogenic bacteria, separation and purification after tieback, sequencing and other processes, which are time-consuming and laborious. Molecular detection technology can make pathogen identification more efficient. Fusarium control mainly includes cultivating disease-resistant varieties, chemical control, physical control, biological control and crop rotation. In recent years, various root rot bacteria caused by Fusarium have shown an increasing trend. By reviewing the previous relevant research results, this study summarized the research progress of molecular detection and prevention measures of root rot caused by the soilborne disease fungus Fusarium, and explored the current research status and existing problems of fusarium. In order to provide theoretical reference for further study of soil-borne pathogenic fungi.

β-Carboline Alkaloids from Peganum harmala Inhibit Fusarium oxysporum from Codonopsis radix through Damaging the Cell Membrane and Inducing ROS Accumulation.

Fusarium oxysporum is a widely distributed soil-borne pathogenic fungus that can cause medicinal herbs and crops to wither or die, resulting in great losses and threat to public health. Due to the emergence of drug-resistance and the decline of the efficacy of antifungal pesticides, there is an urgent need for safe, environmentally friendly, and effective fungicides to control this fungus. Plant-derived natural products are such potential pesticides. Extracts from seeds of Peganum harmala have shown antifungal effects on F. oxysporum but their antifungal mechanism is unclear. In vitro antifungal experiments showed that the total alkaloids extract and all five β-carboline alkaloids (βCs), harmine, harmaline, harmane, harmalol, and harmol, from P. harmala seeds inhibited the growth of F. oxysporum. Among these βCs, harmane had the best antifungal activity with IC50 of 0.050 mg/mL and MIC of 40 μg/mL. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results revealed that the mycelia and spores of F. oxysporum were morphologically deformed and the integrity of cell membranes was disrupted after exposure to harmane. In addition, fluorescence microscopy results suggested that harmane induced the accumulation of ROS and increased the cell death rate. Transcriptomic analysis showed that the most differentially expressed genes (DEGs) of F. oxysporum treated with harmane were enriched in catalytic activity, integral component of membrane, intrinsic component of membrane, and peroxisome, indicating that harmane inhibits F. oxysporum growth possibly through damaging cell membrane and ROS accumulation via regulating steroid biosynthesis and the peroxisome pathway. The findings provide useful insights into the molecular mechanisms of βCs of P. harmala seeds against F. oxysporum and a reference for understanding the application of βCs against F. oxysporum in medicinal herbs and crops.

The efficacy of Iranian Pythium oligandrum isolates in biocontrol of soil-borne fungal pathogens of tomato

The efficacy of Iranian Pythium oligandrum isolates in biocontrol of soil-borne fungal pathogens of tomato

Endophytic Bacillus subtilis antagonize soil-borne fungal pathogens and suppress wilt complex disease in chickpea plants (Cicer arietinum L.).

The present study aimed to identify potential endophytic bacteria antagonistic against three soil-borne fungal pathogens, Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum f.sp. ciceri causing root rot, collar rot, and fungal wilt diseases in chickpea plants, respectively. A total of 255 bacterial endophytes were isolated from the leaves, stems, and roots of seven different crop plants (chickpea, tomato, wheat, berseem, mustard, potato, and green pea). The dual culture-based screening for antifungal properties indicated that three endophytic isolates had strong inhibition (>50%) against all three pathogens tested. Based on morphological, biochemical, and molecular characterization, the selected isolates (TRO4, CLO5, and PLO3) were identified as different strains of Bacillus subtilis. The bacterial endophytes (TRO4 and CLO5) were positive for plant growth promoting (PGP) traits viz., ammonia, siderophore, and indole-3-acetic acid (IAA) production. The bio-efficacy of the endophytes (TRO4, CLO5, and PLO3) was tested by an in planta trial in chickpea pre-challenged with R. solani, S. rolfsii, and F. oxysporum f.sp. ciceri. The B. subtilis strains TRO4 and CLO5 were found to be effective in reducing percent disease incidence (p ≤ 0.05) and enhancing plant growth parameters. The different root parameters viz. root length (mm), surface area (cm2), root diameter (mm), and root volume (cm3) were significantly (p ≤ 0.05) increased in TRO4 and CLO5 inoculated chickpea plants. Confocal Scanning Laser Microscopy showed heavy colonization of bacteria in the roots of endophyte-inoculated chickpea plants. The inoculation of endophytic Bacillus subtilis strains TRO4 and CLO5 in chickpea plants through seed biopriming reduced the accumulation of superoxide, enhanced the plant defense enzymes, and induced the expression of Pathogenesis-Related (PR) genes. Semi-quantitative analysis of defense-related genes showed differential activation of PR genes (60srp and IFR) by endophyte inoculation. The results of the present study reveal the antagonistic potential of B. subtilis strains TRO4 and CLO5 against three major soil-borne fungal pathogens and their ability to suppress wilt complex disease in chickpea plants. This is the first report on the simultaneous suppression of three major soil-borne fungal pathogens causing wilt complex in chickpea plants by endophytic B. subtilis strains.

Efficacy of Bacillus subtilis (Ehrenberg1835) Cohn1872, in suppressing Fusarium oxysporum Schlecht. emend. Snyder & Hansen, the causal agent of root rot of date palm offshoots (Phoenix dactylifera L.) in Iraq

Date palm root rot disease is one of the most important diseases of date palms and offshoots. It is caused by many soil-borne pathogenic fungi. Pathogenicity assays of the isolated fungi showed that the major causative agents of root rot disease in date palm plantlets were Fusarium oxysporum Schlecht. emend. Snyder & Hansen, F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg S1, F. proliferatum S2, Gibberella fujikuroi (Sawada) Wollenw., and Rhizoctonia solani J.G. Kühn. The most virulent fungus was F. oxysporum with a severity index of 82.16 % of root rot, while R. solani was the least harmful with a disease severity rate of 12.42 %. In laboratory tests, Bacillus subtilis reduced the radial mycelial growth of F. oxysporum on PDA medium by 86.6 %. The application of B. subtilis in combination with F. oxysporum substantially inhibited the severity of root rot disease relative to plantlets treated with only F. oxysporum. In addition, B. subtilis application in the presence or absence of F. oxysporum improved the plant physiology of plantlets, including total chlorophyll, total carotenoid, antioxidant enzyme levels (catalase and peroxidase), and total proline content.

The secreted FoAPY1 peptidase promotes Fusarium oxysporum invasion.

The secretion of peptidases from several pathogens has been reported, but the biological function of these proteins in plant-pathogen interactions is poorly understood. Fusarium oxysporum, a soil-borne plant pathogenic fungus that causes Fusarium wilt in its host, can secrete proteins into host plant cells during the infection process to interfere with the host plant defense response and promote disease occurrence. In this study, we identified a peptidase, FoAPY1, that could be secreted from F. oxysporum depending on the N-terminal signal peptide of the protein. FoAPY1 belongs to the peptidase M28 family and exerts peptidase activity in vitro. Furthermore, the FoAYP1 gene knockout strain (∆FoAYP1) presented reduced virulence to tomato plants, but its mycelial growth and conidiation were unchanged. Moreover, FoAYP1 overexpression tomato seedlings exhibited enhanced susceptibility to F. oxysporum and Botrytis cinerea strains. These data demonstrated that FoAYP1 contributes to the virulence of F. oxysporum may through peptidase activity against host plant proteins.

Discovery of Fusarium proliferatum f. sp. malus domestica Causing Apple Replant Disease in China.

Apple replant disease (ARD) is the most serious threat facing the apple industry globally. ARD is mainly manifested as decreased plant growth, serious root rot disease, and considerable yield loss. Microbial factors are the dominant factors leading to the occurrence of ARD. Research on soil-borne pathogenic fungi leading to the occurrence of ARD in China is limited. In the present study, we selected 16 replanting orchards from the Northwest Loess region and around the Bohai Gulf. Diseased roots and rhizosphere soil from healthy apple trees and trees showing ARD symptoms were sampled at random. High-throughput sequencing was used to study the fungal communities in the rhizosphere soil, which showed that the composition of the rhizosphere soil fungal community of ARD-symptomatic and healthy apple trees was different. Nectriaceae at the family level and Fusarium at the genus level dominated the rhizosphere soil fungal community in the two regions, while for healthy apple trees, the relative abundance of Mortierella, Minimedusa, Tetracladium, and Chaetomium was higher. Tissue separation and serial dilution were used to separate fungi, and a total of 89 genera and 219 species were obtained, most of which were Fusarium. Fusarium was further confirmed to be the most abundant pathogen species leading to the occurrence of ARD in China through pathogenicity assays. A pathogenicity assay was carried out by the dip-and-cut technique using different host plants. It was found that Fusarium MR5 showed strong aggressiveness to apple rootstocks. Diseased seedlings specifically exhibited chlorosis of the leaves, browning from the edge of the leaf, followed by rolling and yellowing of the leaves, resulting in wilting and eventually death. Strain MR5 was preliminarily identified as F. proliferatum according to the morphological and cultural characteristics. A maximum likelihood analysis of identities based on six gene sequence (ITS, TUB2, IGS, mtSSU, RPB2, and the TEF gene) alignments between the MR5 strain and other strains showed 99 to 100% homology with F. proliferatum. Based on our test results, strain MR5 was identified as F. proliferatum f. sp. malus domestica, which is of great significance for finding new measures to control ARD in China.

Cover Crops as Reservoirs for Young Vine Decline Pathogens

Young vine decline (YVD) is a grapevine trunk disease (GTD) which results in stunted and delayed growth, reduced yield, root necrosis and eventually death of young vines. Given losses associated with root trunk disease, and increasing limits on chemical fungicides, there is a need for sustainable approaches to combat disease; (1) Cover cropping is a commonly used practice in agricultural systems and has potential to reduce disease in vineyards but there is a risk that cover crop species may act as a host for grapevine pathogens, increasing the risk of infection; (2) We tested 25 plant species commonly used in cover crops to assess their potential to act as a host for a Ilyonectria liriodendri, which is a causal agent of young vine decline. We inoculated greenhouse pots with a pathogeninc strain of Ilyonectria and assayed the roots for the presence of the pathogen; (3) Of the 25 cover crops tested, many of the species showed increased root abundance of Ilyonectria, compared to background levels. In particular phacelia (Phacelia tanacetifolia) and buckwheat (Fagopyrum esculentum) showed very high levels of root colonization. (4) This is the first study to our knowledge that highlights the potential of cover crops to soil borne fungal pathogens.

Leguminous cover crops and soya increased soil fungal diversity and suppressed pathotrophs caused by continuous cereal cropping.

The enrichment of soil-borne fungal pathogens and a high input of mineral fertilizer in the continuous cropping of cereal crops have raised a concern about soil health deterioration. Conversion of continuous cereal cropping to a legume-involved system alters the soil fungal community. However, when a leguminous cover crop is grown with a succeeding legume grain crop such as soya (Glycine max L. Merril), the effects on the soil fungal community when two legumes are involved in the crop system remain unclear. Thus, the effects of the cover crop on the soil fungal community under a succession of soya and a succession of maize (Zea mays L.) were clarified: a continuous wheat (Triticum aestivum L.)–maize cropping system was converted to new rotation systems with three cover crop treatments: leguminous vetch (Vicia sativa L.), a mixture of vetch and rye (Secale cereale L.), and fallow, succeeded by soya or maize in this study. The soil fungal community at the harvest of soya and maize were determined using high-throughput sequencing of ITS2 amplicons. Compared to a wheat–maize rotation system, all of the new rotation systems that involved leguminous crops or fallow increased the soil fungal diversity and suppressed pathotrophs by reducing the soil NH4+, NO3−, available K, and available P concentrations. Different cover crops changed the fungal community composition, but their effect was overwhelmed by the strong effect of succeeding soya, which induced minor shifts among the cover crop treatments under soya than maize. The Vetch–Soya system exhibited the highest fungal diversity, which have been due to an increase of symbiotrophs. Replacing wheat with mixed vetch and rye most greatly suppressed the pathotrophs, and this suppression effect was stronger when succeeded by maize than by soya. These results showed the short-term benefits of legume–legume succession and legume–cereal mixed cover crops for increasing fungal diversity and suppressing pathotrophs. Further study is needed to examine the long-term effects of Vetch–Soya on the accumulation of legume-associated pathogens.

An auxiliary activity family 9 protein, VdAA91, is required for the penetration structure formation in Verticillium dahliae

Verticillium dahliae is a soil-borne fungal pathogen that can cause severe vascular wilt in many plant species; however, the role of some proteins secreted during infection needs to be explored. The fungal genome contains multiple auxiliary activity family 9 (AA9) genes that encode a conserved lytic polysaccharide monooxygenase domain, and some AA9 genes are required for the virulence of plant pathogenic fungi. To investigate the potential roles of AA9 genes, genome-wide identification and characterization were performed. A total of 20 AA9 genes were identified in the V. dahliae genome. According to a gene knockout experiment, VdAA91 is essential for the pathogenicity of V. dahliae. Furthermore, sequence analysis revealed that VdAA91 contains three conserved amino acid residues (His17, His114, and Tyr193) in the AA9 protein family. Subcellular localization and western blotting analyses revealed that VdAA91 is a secretory protein that is localized in the apoplast space of host cells. VdAA91 mutants showed defects in virulence and failures in root colonization based on observation of green fluorescent protein-labeled V. dahliae. We also observed penetration structure and its derived septin neck formation under microscopy. The VdAA91 mutants were incapable of producing penetration pegs and a VdSep5-GFP ring signal. Taken together, our results showed deletion of VdAA91 impairs the formation of the penetration structure and ultimately compromises V. dahliae colonization in the vascular bundles in the xylem of cotton.