High-Quality Nuclear Genome and Mitogenome of Bipolaris sorokiniana LK93, a Devastating Pathogen Causing Wheat Root Rot.

Bipolaris sorokiniana, one of the most devastating hemibiotrophic fungal pathogens, causes root rot, crown rot, leaf blotching, and black embryos of gramineous crops worldwide, posing a serious threat to global food security. However, the host-pathogen interaction mechanism between B. sorokiniana and wheat remains poorly understood. To facilitate related studies, we sequenced and assembled the genome of B. sorokiniana LK93. Nanopore long reads and next generation sequencing short reads were applied in the genome assembly, and the final 36.4-Mb genome assembly contains 16 contigs with the contig N50 of 2.3 Mb. Subsequently, we annotated 11,811 protein-coding genes. Of these, 10,620 were functional genes, 258 of which were identified as secretory proteins, including 211 predicted effectors. Additionally, the 111,581-bp mitogenome of LK93 was assembled and annotated. The LK93 genomes presented in this study will facilitate research in the B. sorokiniana-wheat pathosystem for better control of crop diseases. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pathotype Identification and Virulence Variation in Cochliobolus sativus in China.

Spot blotch caused by Cochliobolus sativus has become an important disease in the wheat-growing regions in China that has resulted from changes in the regional climate, agricultural cultivation patterns, and the susceptible wheat varieties that are widely grown. Little information is available about virulence variability and pathogenic specialization of the C. sativus isolates from major wheat-growing regions in China. Here, 12 representative wheat varieties and foundation breeding stocks were selected to characterize the pathotypes of C. sativus isolates from infected wheat plants. Based on the infection phenotypes in the 12 differential genotypes at the seedling stage, 70 Chinese pathotypes were identified from 110 isolates and clustered into three virulence groups. The high virulence isolates were collected from wheat leaves, crowns, and roots, with most (10 of 14) from the Henan province in the Huang-Huai plain. No relationship was evident between virulence variability of C. sativus isolates and their geographic origins or types of diseased wheat tissues. C. sativus showed a significant pathogenic specialization in hosts of wheat and barley. Most of the wheat isolates (50 of 65) were avirulent to all the differential barley genotypes, and a few were virulent only to highly susceptible barley genotypes. These results indicated that C. sativus isolates from the wheat-growing regions in China varied considerably for their virulence in wheat varieties, and showed significant pathogenic specialization to the wheat and barley hosts.

An arabinose-induced enhancement of asexual reproduction and concomitant changes in metabolic state in the filamentous fungus Bipolaris maydis.

l-Arabinose, a major constituent pentose of plant cell-wall polysaccharides, has been suggested to be a less preferred carbon source for fungi but to be a potential signalling molecule that can cause distinct genome-wide transcriptional changes in fungal cells. Here, we explore the possibility that this unique pentose influences the morphological characteristics of the phytopathogenic fungus Bipolaris maydis strain HITO7711. When grown on plate media under different sugar conditions, the mycelial dry weight of cultures on l-arabinose was as low as that with no sugar, suggesting that l-arabinose does not substantially contribute to vegetative growth. However, the intensity of conidiation on l-arabinose was comparable to or even higher than that on d-glucose and on d-xylose, in contrast to the poor conidiation under the no-sugar condition. To explore the physiological basis of the passive growth and active conidiation on l-arabinose, we next investigated cellular responses of the fungus to these sugar conditions. Transcriptional analysis of genes related to carbohydrate metabolism showed that l-arabinose stimulates carbohydrate utilization through the hexose monophosphate shunt (HMP shunt), a catabolic pathway parallel to glycolysis and which participates in the generation of the reducing agent NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). Then, the HMP shunt was impaired by disrupting the related gene BmZwf1, which encodes glucose-6-phosphate dehydrogenase in this fungus. The resulting mutants on l-arabinose showed remarkably decreased conidiation, but a conversely increased mycelial dry weight compared with the wild-type. Our study demonstrates that l-arabinose acts to enhance resource allocation to asexual reproduction in B. maydis HITO7711 at the cost of vegetative growth, and suggests that this is mediated by the concomitant stimulation of the HMP shunt.

Septins are required for reproductive propagule development and virulence of the maize pathogen Cochliobolus heterostrophus.

Septins are highly conserved GTP-binding proteins that function in cell cytokinesis, polarity and morphogenesis. To evaluate the roles of these proteins in inoculum health and disease, mutants deleted for each of five septin proteins (Cdc3, Cdc10, Cdc11, Cdc12, and Cdc100) were characterized in the ascomycete Cochliobolus heterostrophus for ability to develop asexual and sexual spores and for virulence to the host maize. Strains deleted for CDC3, CDC10, CDC11, and CDC12 genes showed significant changes in hyphal growth, and in development of conidia and ascospores compared to the wild-type strain. Conidia had dramatically reduced numbers of septa and rates of germination, while ascospore development was blocked in the meiotic process. Although asci were produced, wild-type ascospores were not. When equal numbers of conidia from wild type and mutants were used to inoculate maize, cdc10 mutants showed reduced virulence compared to the wild-type strain and other mutants. This reduced virulence was demonstrated to be correlated with lower germination rate of cdc10 mutant conidia. When adjusted for germination rate, virulence was equivalent to the wild-type strain. Double mutants (cdc3cdc10, cdc3cdc11) showed augmented reduced growth phenotypes. cdc100 mutants were wild type in all assays. Taken together, these findings indicate that all four conserved septin proteins play a major role in reproductive propagule formation and that mutants with deletions of CDC10 are reduced in virulence to the host maize.

Engineering of a highly thermostable endoglucanase from the GH7 family of Bipolaris sorokiniana for higher catalytic efficiency.

In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and ß-glucosidase towards complete saccharification of cellulose into glucose.

Genome-based mining of new antimicrobial meroterpenoids from the phytopathogenic fungus Bipolaris sorokiniana strain 11134.

Polyketide-terpenoid hybrid compounds are one of the largest families of meroterpenoids, with great potential for drug development for resistant pathogens. Genome sequence analysis of secondary metabolite gene clusters of a phytopathogenic fungus, Bipolaris sorokiniana 11134, revealed a type I polyketide gene cluster, consisting of highly reducing polyketide synthase, non-reducing polyketide synthase, and adjacent prenyltransferase. MS- and UV-guided isolations led to the isolation of ten meroterpenoids, including two new compounds: 19-dehydroxyl-3-epi-arthripenoid A (1) and 12-keto-cochlioquinone A (2). The structures of 1-10 were elucidated by the analysis of NMR and high-resolution electrospray ionization mass spectroscopy data. Compounds 5-8 and 10 showed moderate activity against common Staphylococcus aureus and methicillin-resistant S. aureus, with minimum inhibitory concentration (MIC) values of 12.5-100 µg/mL. Compound 5 also exhibited activity against four clinical resistant S. aureus strains and synergistic antifungal activity against Candida albicans with MIC values of 12.5-25 µg/mL. The biosynthetic gene cluster of the isolated compounds and their putative biosynthetic pathway are also proposed. KEY POINTS: • Ten meroterpenoids were identified from B. sorokiniana, including two new compounds. • Cochlioquinone B (5) showed activity against MRSA and synergistic activity against C. albicans. • The biosynthetic gene cluster and biosynthetic pathway of meroterpenoids are proposed. • Genome mining provided a new direction to uncover the diversity of meroterpenoids.

Seeing double on Cannabis: Haploids and heteroploids of Bipolaris gigantea on hemp and other dicots.

Bipolaris gigantea (= Drechslera gigantea) causes Bipolaris leaf spot (BLS), a devastating and widespread disease on industrial hemp (Cannabis sativa). An investigation of relationships of isolates from hemp and other plants indicated variation in ploidy that has not previously been reported for Bipolaris. Isolates were obtained from BLS lesions on hemp and nearby weeds in 11 Kentucky counties and were similar to each other in morphology and growth characteristics. In total, 23 isolates were analyzed by multilocus phylogenetics, of which seven were also chosen for whole genome shotgun sequencing. Genes for RNA polymerase II subunit 2 (RPB2), translation elongation factor 1-α (TEF1), and mating type (MAT1) indicated that 13 of the isolates were haploid with only a single allele each of RPB2 and TEF1 and either the MAT1-1 or MAT1-2 idiomorph, whereas 10 were apparently "heteroploid" with two alleles each of RPB2 and TEF1 and both MAT1 idiomorphs. Haploids all had identical RPB2 alleles except for a 1-bp difference in two isolates, identical TEF1 alleles, and (if present) identical MAT1-2 alleles. Those alleles were also present in each heteroploid along with either of two related but distinct alleles for each gene. In contrast, haploids and heteroploids shared allelic variation of MAT1-1. In total, four haploid and two heteroploid genotypes were identified. Genome sequence data assembled to 30-32 Mb for each of four haploid isolates, but 10-31 Mb larger sizes for each of three heteroploids depending on sequencing platform and assembly program. The haploids and heteroploids caused similar disease on hemp.

Phylogeographic Diversity Analysis of Bipolaris sorokiniana (Sacc.) Shoemaker Causing Spot Blotch Disease in Wheat and Barley.

Bipolaris sorokiniana is a fungal pathogen that infects wheat, barley, and other crops, causing spot blotch disease. The disease is most common in humid, warm, wheat-growing regions, with South Asia's Eastern Gangetic Plains serving as a hotspot. There is very little information known about its genetic variability, demography, and divergence period. The current work is the first to study the phylogeographic patterns of B. sorokiniana isolates obtained from various wheat and barley-growing regions throughout the world, with the goal of elucidating the demographic history and estimating divergence times. In this study, 162 ITS sequences, 18 GAPDH sequences, and 74 TEF-1α sequences from B. sorokiniana obtained from the GenBank, including 21 ITS sequences produced in this study, were used to analyse the phylogeographic pattern of distribution and evolution of B. sorokiniana infecting wheat and barley. The degrees of differentiation among B. sorokiniana sequences from eighteen countries imply the presence of a broad and geographically undifferentiated global population. The study provided forty haplotypes. The H_1 haplotype was identified to be the ancestral haplotype, followed by H_29 and H_27, with H_1 occupying a central position in the median-joining network and being shared by several populations from different continents. The phylogeographic patterns of species based on multi-gene analysis, as well as the predominance of a single haplotype, suggested that human-mediated dispersal may have played a significant role in shaping this pathogen's population. According to divergence time analysis, haplogroups began at the Plio/Pleistocene boundary.

Molecular diversity, haplotype distribution and genetic variation flow of Bipolaris sorokiniana fungus causing spot blotch disease in different wheat-growing zones.

Bipolaris sorokiniana (BS) is an economically important fungal pathogen causing spot blotch of wheat (Trtiticum aestivum) and found in all wheat-growing zones of India. Very scanty and fragmentary information is available on its genetic diversity. The current research is the first detailed report on the geographic distribution and evolution of BS population in five geographically distinct wheat-growing zones (North Western Plain Zone (NWPZ), North Eastern Plain zone (NEPZ), North Hill Zone (NHZ), Southern Hill Zone (SHZ) and Peninsular Zone (PZ)) of India, studied by performing nucleotide sequence comparison of internal transcribed spacer region of 528 isolates. A moderate to low levels of haplotypic diversity was noticed in different wheat-growing zones. Phylogenetic analysis suggests that B. sorokiniana exist in two distinct lineages as all isolates under study were grouped in two different clades and found analogous to the findings of haplotypic and TCS network analysis. The genetic parameters revealed the existence of 40 haplotypes with three major haplotypes (H-1, H-2 and H-3) which showed star-like structure network surrounded by several single haplotypes, revealing high frequency of the mutations (Eta = 2 - 158) in total analyzed population. H-1 was observed as a predominant haplotype and prevalent in all the five zones. Moderate level of genetic differentiation was found between NHZ and other zones like NWPZ (Fst = 0.332) and SHZ (Fst = 0.382) and PZ (Fst = 0.299), whereas it was low between NEPZ and PZ (Fst = 0.034). Higher transfer rate of genetic variation was noticed between NEPZ and PZ (Nm = 7.06), while it was found minimum between NHZ and SHZ (Nm = 0.40). Moreover, negative score of neutrality statistics (Tajima's D and Fu's FS test) for NWPZ population suggested recent population expansion. However, positive score for both the neutrality tests observed in NEPZ indicated the dominance of balancing selection in structuring their population. Recombination events were observed in the NWPZ and NHZ population, while it was absent in SHZ, NEPZ and PZ population. Thus, the lack of any specific genetic population structure in all the zones indicates for the expansion history only from one common source population, i.e. NWPZ, a mega zone of wheat production in India. Overall, it seems that the predominance of individual haplotypes with a moderate level of genetic variation and human-mediated movement of contaminated seed and dispersal of inoculum, mutations and recombination as prime evolutionary processes play essential role in defining the genetic structure of BS population.

Setosphaeria turcica ATR turns off appressorium-mediated maize infection and triggers melanin-involved self-protection in response to genotoxic stress.

Eukaryotic organisms activate conserved signalling networks to maintain genomic stability in response to DNA genotoxic stresses. However, the coordination of this response pathway in fungal pathogens remains largely unknown. In the present study, we investigated the mechanism by which the northern corn leaf blight pathogen Setosphaeria turcica controls maize infection and activates self-protection pathways in response to DNA genotoxic insults. Appressorium-mediated maize infection by S. turcica was blocked by the S-phase checkpoint. This repression was dependent on the checkpoint central kinase Ataxia Telangiectasia and Rad3 related (ATR), as inhibition of ATR activity or knockdown of the ATR gene recovered appressorium formation in the presence of genotoxic reagents. ATR promoted melanin biosynthesis in S. turcica as a defence response to stress. The melanin biosynthesis genes StPKS and StLac2 were induced by the ATR-mediated S-phase checkpoint. The responses to DNA genotoxic stress were conserved in a wide range of phytopathogenic fungi, including Cochliobolus heterostrophus, Cochliobolus carbonum, Alternaria solani, and Alternaria kikuchiana, which are known causal agents for plant diseases. We propose that in response to genotoxic stress, phytopathogenic fungi including S. turcica activate an ATR-dependent pathway to suppress appressorium-mediated infection and induce melanin-related self-protection in addition to conserved responses in eukaryotes.

Large-spored Drechslera gigantea is a Bipolaris species causing disease on the invasive grass Microstegium vimineum.

Environmentally damaging invasive plants can also serve as reservoir hosts for agricultural pathogens. Microstegium vimineum is an invasive C4 annual grass that is present throughout the midwestern and eastern United States. It can reach high densities in disturbed areas such as crop-forest interfaces, which creates the potential for pathogen spillover from M. vimineum to agricultural crops and native plants. A previous study that surveyed disease on M. vimineum found a large-spored Bipolaris species that was widespread on M. vimineum and also isolated from co-occurring native grasses. Here, we report that the large-spored fungus isolated from M. vimineum and the native grass Elymus virginicus is Drechslera gigantea, based on comparison with published descriptions of morphological traits, and establish that D. gigantea is a pathogen of M. vimineum and E. virginicus. We review the phylogenetic placement and taxonomic history of D. gigantea and propose that it be reassigned to the genus Bipolaris as Bipolaris gigantea.