A novel member of miR169 family negatively regulates maize resistance against Bipolaris maydis.

MicroRNAs (miRNAs) have been confirmed to play important roles in plant defense response. However, the key maize miRNAs involved in the defense response against Bipolaris maydis are very limited. In this study, a novel member of the miR169 family in response to B. maydis, named zma-miR169s, was discovered and investigated. The expression levels of pre-miR169s and zma-miR169s were significantly repressed during B. maydis infection. CRISPR/Cas9-induced zma-miR169s mutant exhibited more resistance against B. maydis, whereas overexpression zma-miR169s enhanced susceptibility, supporting that zma-miR169s might play a negative role in maize resistance. Moreover, RNA-seq and GO analysis showed that differentially expressed genes were highly enriched in the oxidation-reduction process and plant hormone pathway. Hence, reactive oxygen species (ROS) and plant hormone levels were further investigated. ROS detection confirmed that zma-miR169s mutant accumulated more ROS, while less ROS was detected in transgenic maize OE-miR169s. Furthermore, more remarkable changes in PR-1 expression levels and salicylic acid (SA) contents were detected in zma-miR169s mutant compared to wild-type and transgenic maize during B. maydis infection. Additionally, nuclear transcription factors (NF-YA1 and NF-YA13) were identified as targets regulated by zma-miR169s through the agrobacterium-mediated transient expression method. Overexpression of ZmNF-YA13 enhanced Arabidopsis resistance to Pseudomonas syringae pv. tomato DC3000. Taken together, our results suggest that zma-miR169s negatively regulate maize defense responses by influencing ROS accumulation and the SA-dependent signaling pathway.

Investigation on comparative transcriptome profiling of resistant and susceptible non-CMS maize genotypes during Bipolaris maydis race O infection.

Maydis leaf blight is a significant disease of maize caused by Bipolaris maydis race T, O and C. Molecular mechanisms regulating defense responses in non-CMS maize towards race O fungus are not fully known. In the present investigation, comparative transcriptome profiling was conducted on a highly resistant maize genotype SC-7-2-1-2-6-1 against a standard susceptible variety CM 119 at 48 h post inoculation (h PI) along with non-infected control. mRNA sequencing generated 38.4 Gb data, where 9349602 reads were mapped uniquely in SC-7, whereas 2714725 reads were mapped uniquely in CM-119. In inoculated SC-7, the total number of differentially expressed genes (DEGs) against control was 1413, where 1011 were up-regulated, and 402 were down-regulated. In susceptible inoculated genotype CM 119, the number of DEGs against control was 2902, where 1703 were up-, and 1199 were down-regulated. DEGs between inoculated resistant and susceptible genotypes were 10745, where 5343 were up-, and 5402 were down-regulated. The RNA-seq data were validated using RT-qPCR. The key findings are that SC-7 poses a robust plant signaling system mainly induced by oxidation-reduction process and calcium-mediated signaling. It regulates its fitness-related genes efficiently, viz., aldolase 2 gene, isopropanoid, phyto hormones, P450 cytochrome, amino acid synthesis, nitrogen assimilation genes etc. These findings showed more transcriptional changes in the SC-7 genotype, which contains many defence-related genes. They can be explored in future crop development programmes to combat multiple maize diseases. The current finding provides information to elucidate molecular and cellular processes occurring in maize during B. maydis race O infection.

Bipoladien A, a Sesterterpenoid Containing an Undescribed 5/8/5/7 Carbon Skeleton from Bipolaris maydis.

Bipoladiens A-E (1-5), five new ophiobolin-derived sesterterpenoids, and a known compound 6 (bipolaricin R) were isolated from the cultures of the phytopathogenic fungus Bipolaris maydis. Their structures and absolute configurations were elucidated based on comprehensive spectroscopic analyses, HRESIMS, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analyses. Notably, compound 1 has an undescribed tetracyclic 5/8/5/7 fused carbon skeleton, and compound 2 possesses a rare multicyclic caged ring system. The biosynthetic pathway of 1 was proposed starting from 6 via a series of oxidation and cyclization reactions. Compound 6 showed excellent antiproliferation and apoptosis induction effects against A549 cell line. Additionally, compounds 5 and 6 exhibited noticeable antimicrobial ability against Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermidis. These findings not only developed the chemical and bioactivities diversities of ophiobolin-sesterterpenoid but also provided an idea to boost the application of natural products in the control of food pathogens.

The Effects of Mycovirus BmPV36 on the Cell Structure and Transcription of Bipolaris maydis.

Bipolaris maydis partitivirus 36 (BmPV36) is a mycovirus that can significantly reduce the virulence of the host Bipolaris maydis, but its hypovirulence mechanism is not clear. To investigate the response of B. maydis to BmPV36, the effects of BmPV36 on host cell structure and gene expression were studied via transmission electron microscopy and transcriptome sequencing using BmPV36-carrying and virus-free mycelium on the second and fifth culture. The results of transmission electron microscopy showed that the cell wall microfibrils of B. maydis were shortened, the cell membrane was broken, and membrane-bound vesicles and vacuoles appeared in the cells after carrying BmPV36. Transcriptome sequencing results showed that after carrying BmPV36, B. maydis membrane-related genes were significantly up-regulated, but membrane transport-related genes were significantly down-regulated. Genes related to carbohydrate macromolecule polysaccharide metabolic and catabolic processes were significantly down-regulated, as were genes related to the synthesis of toxins and cell wall degrading enzymes. Therefore, we speculated that BmPV36 reduces the virulence of B. maydis by destroying the host's cell structure, inhibiting the synthesis of toxins and cell wall degrading enzymes, and reducing cell metabolism. Gaining insights into the hypovirulence mechanism of mycoviruses will provide environmentally friendly strategies for the control of fungal diseases.

Four closely related endornaviruses each with a low incidence in the phytopathogenic fungi Exserohilum turcicum or Bipolaris maydis.

Endornaviruses are known to occur widely in plants, fungi, and oomycetes, but our understanding of their diversity and distribution is limited. In this study, we report the discovery of four endornaviruses tentatively named Setosphaeria turcica endornavirus 1 (StEV1), Setosphaeria turcica endornavirus 2 (StEV2), Bipolaris maydis endornavirus 1 (BmEV1), and Bipolaris maydis endornavirus 2 (BmEV2). StEV1 and StEV2 infect Exserohilum turcicum, while BmEV1 and BmEV2 infect Bipolaris maydis. The four viruses encode a polyprotein with less than 40 % amino acid sequence identity to other known endornaviruses, indicating that they are novel, previously undescribed endornaviruses. However, StEV1 and BmEV1 share a sequence identity of 78 % at the full-genome level and 87 % at the polyprotein level, suggesting that they may belong to the same species. Our study also found that each of the four endornaviruses has an incidence of approximately 3.5 % to 5.5 % in E. turcicum or B. maydis. Interestingly, BmEV1 and BmEV2 were found to be unable to transmit between hosts of different vegetative incompatibility groups, which may explain their low incidence.

Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Abstract Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Resumo A ferrugem das folhas de maydis, causada por Bipolaris maydis, é uma doença importante da cultura do milho em Khyber Pakhtunkhwa (KP), Paquistão. Quinze isolados do patógeno, coletados em KP, foram estudados quanto à variabilidade com base em marcadores fenotípicos e moleculares. Variabilidade significativa entre os isolados foi observada quando avaliada por meio de características fenotípicas, como crescimento radial, concentração de esporos, sensibilidade a fungicida e virulência. Os isolados foram classificados em seis grupos de cultura com base na cor, textura e margens da colônia. A morfologia dos conídios também foi variável. Estes eram retos ou ligeiramente curvos e de cor marrom-claro a escuro. O teste de fungicida mostrou variação significativa no grau de sensibilidade ao carbendazim. O isolado Bm8 exibiu crescimento radial máximo em placas com adição de carbendazim. Por outro lado, o isolado Bm15 apresentou o menor crescimento radial. As variações no padrão de virulência dos isolados foram evidentes quando uma variedade de milho suscetível Azam foi inoculada com esporos de B. maydis. A variabilidade genética entre os isolados também foi estimada por RAPD, bem como sequenciamento da região ITS. O dendrograma RAPD agrupou todos os isolados em dois grupos principais. A distância genética média variou de 0,6% a 100%, indicando uma lacuna genética diversa entre os isolados. A distância genética máxima foi encontrada entre os isolados Bm9 e Bm10 e também entre Bm2 e Bm8. Por outro lado, os isolados Bm13 e Bm15 estavam a uma distância genética mínima. O dendrograma filogenético baseado no sequenciamento da região ITS agrupou todos os isolados em um único aglomerado principal. Os agrupamentos em ambos os dendrogramas não se correlacionam com a distribuição geográfica nem com as características morfológicas.

Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens.

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens-mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum, responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions.

Population structure and mixed reproductive strategies in Bipolaris maydis from single and multiple corn cultivars in Fujian Province, China.

Bipolaris maydis is the pathogenic microorganism of southern corn leaf blight, a persistent biotic constraint responsible for substantial yield losses of corn worldwide. In the present study, 96 isolates from six representative fields growing single and multiple sweet corn cultivars in Pingnan, Fuqing, and Jian'ou in Fujian Province, which are characterized by different geographical characteristics and cropping patterns, were genetically analyzed using inter-simple sequence repeat (ISSR) markers to assess the impact of geographical origins and corn cultivars on B. maydis population differentiation. B. maydis isolates originated from diverse regions possessed higher genetic variety than those from single and multiple sweet corn cultivars. Phylogenetic analysis showed that the isolates from single and multiple sweet corn cultivars were randomly grouped into different clusters, with those from the same location tending to form clusters. A greater genetic differentiation among different geographical populations than between those from single and multiple sweet corn cultivars was observed by pairwise comparison. Hierarchical analysis indicated that among-population variation was higher when comparatively analyzed B. maydis populations from different locations than in those from single and multiple sweet corn cultivars. In conclusion, these results suggest that geographical origin acts a more considerable role in genetic differentiation of B. maydis than corn cultivar. Two divided genetic clusters were detected in the B. maydis populations from single and multiple sweet corn cultivars at the three locations in Fujian Province, with major genetic variation being derived within populations. The high haplotypic diversity and expected mating type ratio of 1:1 in combination with significant linkage disequilibrium suggested that a mixed reproductive strategy occurs in the B. maydis population in Fujian Province. This study will enrich the information on the role that geographical origins and corn cultivars play in the population structure of the pathogen as well as the reproductive strategies in B. maydis population in Fujian Province.

New Lactones Produced by Streptomyces sp. SN5431 and Their Antifungal Activity against Bipolaris maydis.

Bipolaris maydis causes southern corn leaf blight and inflicts huge losses on maize production. In order to search for new natural products from insect gut bacteria to control plant fungal disease, 86 actinomycetes were isolated from more than 50 insect guts, in which crude extract of strain SN5431 showed significant inhibition of the mycelial growth of B. maydis. The strain was identified and named as Streptomyces sp. SN5431. Six compounds were obtained from the crude extract of strain SN5431, which includes five new γ-butyrolactones named as tiuslactone A-E (1-5), and one new long chain ester named as tiusester (6). Their structures were determined using NMR and HRESIMS data and then combined with the spectroscopic data of known similar compounds. Tiuslactone B (2) showed powerful antifungal activity against B. maydis. These results indicated metabolites of insect gut bacteria have the potential to be the leading compounds for the control of corn leaf blight.

Peptidome and Transcriptome Analysis of Plant Peptides Involved in Bipolaris maydis Infection of Maize.

Southern corn leaf blight (SCLB) caused by Bipolaris maydis threatens maize growth and yield worldwide. In this study, TMT-labeled comparative peptidomic analysis was established between infected and uninfected maize leaf samples using liquid-chromatography-coupled tandem mass spectrometry. The results were further compared and integrated with transcriptome data under the same experimental conditions. Plant peptidomic analysis identified 455 and 502 differentially expressed peptides (DEPs) in infected maize leaves on day 1 and day 5, respectively. A total of 262 common DEPs were identified in both cases. Bioinformatic analysis indicated that the precursor proteins of DEPs are associated with many pathways generated by SCLB-induced pathological changes. The expression profiles of plant peptides and genes in maize plants were considerably altered after B. maydis infection. These findings provide new insights into the molecular mechanisms of SCLB pathogenesis and offer a basis for the development of maize genotypes with SCLB resistance.

Investigation of the antifungal activity of the dicarboximide fungicide iprodione against Bipolaris maydis.

Southern corn leaf blight (SCLB), mainly caused by Bipolaris maydis, is a destructive disease of maize worldwide. Iprodione is a widely used dicarboximide fungicide (DCF); however, its antifungal activity against B. maydis has not been well studied until now. In this study, the sensitivity of 103 B. maydis isolates to iprodione was determined, followed by biochemistry and physiology assays to ascertain the fungicide's effect on the morphology and other biological properties of B. maydis. The results indicated that iprodione exhibited strong inhibitory activity against B. maydis, and the EC50 values in inhibiting mycelial growth ranged from 0.088 to 1.712 µg/mL, with a mean value of 0.685 ± 0.687 µg/mL. After treatment with iprodione, conidial production of B. maydis was decreased significantly, and the mycelia branches increased with obvious shrinkage, distortion and fracture. Moreover, the expression levels of the osmotic pressure-related regulation genes histidine kinase (hk) and Ssk2-type mitogen-activated protein kinase (ssk2) were upregulated, the glycerin content of mycelia increased significantly, the relative conductivity of mycelia increased, and the cell wall membrane integrity was destroyed. The in vivo assay showed that iprodione at 200 µg/mL provided 79.16% protective efficacy and 90.92% curative efficacy, suggesting that the curative effect was better than the protective effect. All these results proved that iprodione exhibited strong inhibitory activity against B. maydis and provided excellent efficacy in controlling SCLB, indicating that iprodione could be an alternative candidate for the control of SCLB in China.

Multi-Gene Phylogenetic Approach for Identification and Diversity Analysis of Bipolaris maydis and Curvularia lunata Isolates Causing Foliar Blight of Zea mays.

Bipolaris species are known to be important plant pathogens that commonly cause leaf spot, root rot, and seedling blight in a wide range of hosts worldwide. In 2017, complex symptomatic cases of maydis leaf blight (caused by Bipolaris maydis) and maize leaf spot (caused by Curvularia lunata) have become increasingly significant in the main maize-growing regions of India. A total of 186 samples of maydis leaf blight and 129 maize leaf spot samples were collected, in 2017, from 20 sampling sites in the main maize-growing regions of India to explore the diversity and identity of this pathogenic causal agent. A total of 77 Bipolaris maydis isolates and 74 Curvularia lunata isolates were screened based on morphological and molecular characterization and phylogenetic analysis based on ribosomal markers-nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region, 28S nuclear ribosomal large subunit rRNA gene (LSU), D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA), and protein-coding gene-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Due to a dearth of molecular data from ex-type cultures, the use of few gene regions for species resolution, and overlapping morphological features, species recognition in Bipolaris has proven difficult. The present study used the multi-gene phylogenetic approach for proper identification and diversity of geographically distributed B. maydis and C. lunata isolates in Indian settings and provides useful insight into and explanation of its quantitative findings.

Comparative Transcriptome Analysis of Fungal Pathogen Bipolaris maydis to Understand Pathogenicity Behavior on Resistant and Susceptible Non-CMS Maize Genotypes.

Bipolaris maydis is pathogen of maize which causes maydis leaf blight disease. In India major losses occur due to the B. maydis race "O" pathogen, whereas in other parts of the world, major losses are due to the race "T" pathogen. In the present study, we conducted an in planta transcriptomics study of the B. maydis race "O" pathogen after infection on non-CMS maize resistant and susceptible genotypes by mRNA sequencing to understand the molecular basis of pathogenicity for better management of the pathogen. Approximately 23.4 GB of mRNA-seq data of B. maydis were obtained from both resistant and susceptible maize backgrounds for fungus. Differentially expressed genes (DEGs) analysis of B. maydis in two different genetic backgrounds suggested that the majority of highly DEGs were associated with mitochondrial, cell wall and chitin synthesis, sugar metabolism, peroxidase activity, mitogen-activated protein kinase (MAPK) activity, and shikimate dehydrogenase. KEGG analysis showed that the biosynthetic pathways for secondary metabolism, antibiotics, and carbon metabolism of fungus were highly enriched, respectively, in susceptible backgrounds during infection. Previous studies in other host pathogen systems suggest that these genes play a vital role in causing disease in their host plants. Our study is probably the first transcriptome study of the B. maydis race "O" pathogen and provides in-depth insight of pathogenicity on the host.

Maydisone, a novel oxime polyketide from the cultures of Bipolaris maydis.

A novel oxime polyketide, maydisone (1), along with two known compounds, 7-hydroxy-2,5-dimethylchromone (2) and 2,5-dimethylbenzoic acid (3) were isolated from the cultures of Bipolaris maydis. Their structures were identified by the application of NMR and MS data analyses and comparison with previous reports. Compound 1 showed the most powerful inhibition of α-glucosidase, with an IC50 value of 68.30 ± 0.83 µM.

Meiotic Silencing in Dothideomycetous Bipolaris maydis.

The filamentous ascomycete Bipolaris maydis is a plant pathogen that causes corn leaf blight and has been used in cytological studies of sexual reproduction. In this fungus, when null mutants of each septin are crossed with the wild-type strain, all ascospores derived from the same asci show abnormal morphology. The phenomenon was remarkably similar to the event known as "ascus dominance" in Neurospora crassa, which is known to be caused by MSUD (meiotic silencing by unpaired DNA). However, it is not clear whether B. maydis possesses functional MSUD. The object of this study is to elucidate whether this fungus carries a functional MSUD system that causes ascus dominance in the crosses of septin mutants and the wild-type strain. The results of homozygous and heterozygous crossing tests with mutants, having the insertional CDC10-septin gene sequence into the genome, suggested that the ascus dominance in B. maydis is triggered by the unpaired DNA as in N. crassa. To investigate whether MSUD is caused by the same mechanism as in N. crassa, an RNA-dependent RNA polymerase, one of the essential factors in MSUD, was identified and disrupted (Δrdr1) in B. maydis. When the Δrdr1 strain was crossed with each mutant of the septins, ascus dominance did not occur in all crosses. These results suggest that this ascus dominance is caused by RNA silencing triggered by an unpaired gene, as in N. crassa, and septin genes were affected by this silencing. To date, although MSUD has been found only in Fusarium graminearum and N. crassa, which are classified as Sordariomycetes, this study showed that MSUD is also functional in B. maydis, which is classified as a Dothideomycete. These results showed the possibility that this posttranscriptional regulation is extensively conserved among filamentous ascomycetes.

Four distinct isolates of Helminthosporium victoriae virus 190S identified from Bipolaris maydis.

Helminthosporium victoriae virus 190S (HvV190S) is the type species of the genus Victorivirus under the family Totiviridae. To date, HvV190S has never been found in places outside of the USA and has Helminthosporium victoriae as its only know natural host fungus in the field. Here, we report the identification of 4 double-stranded RNA (dsRNA) viruses from Bipolaris maydis in Hubei province of China. Interestingly, the genomes of the 4 viruses show 81.2 %-85.5 % nucleotide sequence identities to HvV190S. Their capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) share 95.5-97.9 % and 94.6-96.6 % amino acid sequence identities to corresponding proteins of HvV190S. Therefore, the 4 viruses, which show 81.8-87.3 % pairwise genome sequence identities, should be considered as distinct isolates of HvV190S. Our finding suggests that HvV190S is widely distributed in the world and may infect fungal species other than H. victoriae.

New secondary metabolites with immunosuppressive activity from the phytopathogenic fungus Bipolaris maydis.

Three previously undescribed compounds, including a meroterpenoid, guignardone T (1), and two ophiobolin-type sesterterpenoids, maydispenoids A and B (2 and 3), along with four known compounds (4-7), were isolated from the phytopathogenic fungus Bipolaris maydis collected from Anoectochilus roxburghii (Wall.) Lindl leaves. The structures of all undescribed compounds were elucidated by spectroscopic analysis, electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction. Structurally, maydispenoids A was characterized by a fascinating decahydro-3-oxacycloocta[cd]pentalene fragment. It is notable that the compounds 2 and 3 exhibited potential inhibitory activity in anti-CD3/anti-CD28 monoclonal antibodies (mAbs) stimulated murine splenocytes proliferation, with IC50 values of 5.28 and 9.38 µM, respectively, and also suppress the murine splenocytes proliferation activated by lipopolysaccharide (LPS), with IC50 values of 7.25 and 16.82 µM, respectively. This is the first report of ophiobolin-type sesterterpenoids as immunosuppressor, and may provide new chemical templates for the development of new immunosuppressive drugs for autoimmune disease treatment.

Characterization of Natural Isolates of Bipolaris maydis Associated with Mating Types, Genetic Diversity, and Pathogenicity in Fujian Province, China.

Due to the natural destructiveness and persistence of the southern corn leaf blight (SCLB) fungus Bipolaris maydis (Nisikado et Miyake) Shoem, the characterization of B. maydis field isolates is essential to guide the rational distribution of resistant materials in corn-growing regions. In the present study, 102 field isolates collected from seven locations covering the entire region of Fujian Province, China, were assessed for mating type distribution, genetic diversity, and pathogenicity toward local sweet corn cultivars. Mating type detection via polymerase chain reaction indicated that 36.3 and 63.7% of isolates were MAT1-1 and MAT1-2, respectively; more than 80% of these isolates were confirmed using cross assays with known mating type isolates. Thirteen intersimple sequence repeat (ISSR) markers within and among two mating type populations revealed a high level of DNA polymorphism for all combined isolates and between MAT1-1 and MAT1-2 populations. The MAT1-2 population was more diverse based on DNA polymorphism than the MAT1-1 population. The value of GST was 0.0070, ranging from 0.0399 to 0.3044 based on analysis of combined isolates and individual regional populations, respectively, suggesting the presence of genetic differentiation in the two mating type populations from different locations. Pathogenicity assays revealed that both MAT1-1 and MAT1-2 populations were pathogenic to all 11 local sweet corn cultivars tested in this study. The potential of sexual reproduction, existence of genetic diversity in the two mating type populations, and pathogenicity suggest that B. maydis populations have independently clonally adapted under natural field conditions during corn cultivation.

Multiplex polymerase chain reaction detection of Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected maize leaf tissues.

Multiplex polymerase chain reaction (PCR) is a method for simultaneous identification and detection of multiple pathogenic fungi, however, its complexity limits its application. To simplify the protocol and to improve the effectiveness, three-level designs for six factors (three primers, Taq DNA polymerase, dNTP, Mg2+ ) were constructed to optimize the multiplex PCR system by using the orthogonal design method and the annealing temperature of the PCR reactions was also optimized. Finally, a multiplex PCR system for the simultaneous detection of these three pathogens of maize was successfully established. The reaction volume was 25 µl and the annealing temperature was 57℃. The optimal conditions for multiplex PCR reaction contained 0.48 µmol/L Cl-1/Cl-2, 0.72 µmol/L Bm-1/Bm-2, 0.24 µmol/L Az-1/Az-2, 1.5 U polymerase, 0.35 mmol/L dNTP, and 1.25 mmol/L MgCl2 . The multiplex PCR system can detect Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected plant tissues rapidly with the sensitivity at 10 pg DNA/µl.