Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

The occurrence and mechanism of field resistance to boscalid and pyraclostrobin in Stemphylium solani, the causal agent of tomato gray leaf spot in China.

The destructive disease gray leaf spot, caused by Stemphylium solani, is prevalent in tomato plants in China. A variety of fungicides have been extensively used for controlling the disease, with a particular focus on succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, there was a lack of information regarding the resistance of S. solani to boscalid (SDHI) and pyraclostrobin (QoI) in China. In this study, the sensitivity of S. solani to boscalid and pyraclostrobin was monitored. The EC50 values for boscalid ranged from 0.02 to 3.0 µg∙mL-1, with an average value of 0.62 µg∙mL-1, while the EC50 values for pyraclostrobin ranged from 0.21 to 14.71 µg∙mL-1, with an average value of 6.03 µg∙mL-1. Based on these findings, the frequencies of observed resistance were as follows: 36.7% for boscalid and 50% for pyraclostrobin; while the resistance frequency to both boscalid and pyraclostrobin in S. solani was 19.4%. The mutation associated with boscalid resistance in S. solani within tomato fields was identified as SdhB-H277Y, while the mutation related to pyraclostrobin resistance was found in cytochrome b, specifically Cytb-G143A. The resistant mutants displayed diminished fitness in terms of mycelial growth, yet their pathogenicity exhibited no significant disparities. To delay the development of resistance, it is advisable to employ a rotation strategy using alternative fungicides with different modes of action or mix with fungicides with multi-site-contact activity for disease management.

Cercospora polygonatum, a New Species Causing Gray Leaf Spot Disease in Polygonatum cyrtonema.

This study identified a new species (Cercospora polygonatum) that causes gray leaf spot (GLS) disease in cultivated Polygonatum cyrtonema. This fungal species was isolated from the affected region of GLS on P. cyrtonema leaves. Pathogenicity bioassays were conducted based on Koch's postulates. Morphology was examined based on the features of conidiomata, conidiogenous loci, conidia/conidiophores, and conidiogenous cells. The rDNA internal transcribed spacer region, calmodulin, translation elongation factor 1-alpha, and histone genes were subjected to phylogenetic analysis using the MrBayes tool in Phylosuite. Bootstrap support analysis for phylogenetic placement confirmed the new species, which was significantly different from the closely related species C. senecionis-walkeri and C. zeae-maydis. The morphological characteristics also supported this finding, with the conidiogenous cells of C. polygonatum being considerably shorter than those of C. senecionis-walkeri or C. zeae-maydis. In addition, C. polygonatum was distinguished by its cultural characteristics. As this fungus was isolated from P. cyrtonema, it was named C. polygonatum F.Q. Yin, M. Liu & W.L. Ma, sp. nov. The type specimen (H8-2) was preserved at the China General Microbiological Culture Collection Center. This is the first report of GLS caused by C. polygonatum on P. cyrtonema leaves in China. The current study enriches the knowledge regarding Cercospora sp., contributes to the identification of a species causing GLS in P. cyrtonema, and provides useful information for the effective management of this disease.

Novel Pestalotiopsis that Causes Gray Spot Disease of Polygonatum cyrtonema in Hunan Province of China.

Polygonatum cyrtonema Hua is a perennial herb of the Asparagaceae family that is used for both dietary and medicinal purposes in China. In September 2019, a new leaf spot disease on Polygonatum cyrtonema was detected and is currently widespread in Huaihua, Hunan Province, China. Pathogenic fungi were isolated and purified from samples of diseased tissue that were collected for morphological and molecular phylogenetic studies. The pathogen was identified using multilocus (ITS, TEF-1, and TUB2) phylogenies, as well as morphological characters, and was found to be clustered but separately divergent from species of Pestalotiopsis. However, there were significant morphological differences between the pathogen and similar species. The pathogen was finally identified as a new species that was designated Pestalotiopsis xuefengensis. This is the first report of Pestalotiopsis xuefengensis serving as the causal agent of gray leaf spot on Polygonatum cyrtonema. This study will provide useful information for the diagnosis and management of this disease.

Integrated transcriptome and DNA methylome analysis reveal the biological base of increased resistance to gray leaf spot and growth inhibition in interspecific grafted tomato scions.

BACKGROUND: Grafting is widely used as an important agronomic approach to deal with environmental stresses. However, the molecular mechanism of grafted tomato scions in response to biotic stress and growth regulation has yet to be fully understood. RESULTS: This study investigated the resistance and growth performance of tomato scions grafted onto various rootstocks. A scion from a gray leaf spot-susceptible tomato cultivar was grafted onto tomato, eggplant, and pepper rootstocks, creating three grafting combinations: one self-grafting of tomato/tomato (TT), and two interspecific graftings, namely tomato/eggplant (TE) and tomato/pepper (TP). The study utilized transcriptome and DNA methylome analyses to explore the regulatory mechanisms behind the resistance and growth traits in the interspecific graftings. Results indicated that interspecific grafting significantly enhanced resistance to gray leaf spot and improved fruit quality, though fruit yield was decreased compared to self-grafting. Transcriptome analysis demonstrated that, compared to self-grafting, interspecific graftings triggered stronger wounding response and endogenous immune pathways, while restricting genes related to cell cycle pathways, especially in the TP grafting. Methylome data revealed that the TP grafting had more hypermethylated regions at CHG (H = A, C, or T) and CHH sites than the TT grafting. Furthermore, the TP grafting exhibited increased methylation levels in cell cycle related genes, such as DNA primase and ligase, while several genes related to defense kinases showed decreased methylation levels. Notably, several kinase transcripts were also confirmed among the rootstock-specific mobile transcripts. CONCLUSIONS: The study concludes that interspecific grafting alters gene methylation patterns, thereby activating defense responses and inhibiting the cell cycle in tomato scions. This mechanism is crucial in enhancing resistance to gray leaf spot and reducing growth in grafted tomato scions. These findings offer new insights into the genetic and epigenetic contributions to agronomic trait improvements through interspecific grafting.

Genetic dissection of resistance to gray leaf spot by genome-wide association study in a multi-parent maize population.

BACKGROUND: Understanding the genetic mechanisms underlying gray leaf spot (GLS) resistance in maize is crucial for breeding GLS-resistant inbred lines and commercial hybrids. Genome-wide association studies (GWAS) and gene functional annotation are valuable methods for identifying potential SNPs (single nucleotide polymorphism) and candidate genes associated with GLS resistance in maize. RESULTS: In this study, a total of 757 lines from five recombinant inbred line (RIL) populations of maize at the F7 generation were used to construct an association mapping panel. SNPs obtained through genotyping-by-sequencing (GBS) were used to perform GWAS for GLS resistance using a linear mixture model in GEMMA. Candidate gene screening was performed by analyzing the 10 kb region upstream and downstream of the significantly associated SNPs linked to GLS resistance. Through GWAS analysis of multi-location phenotypic data, we identified ten candidate genes that were consistently detected in two locations or from one location along with best linear unbiased estimates (BLUE). One of these candidate genes, Zm00001d003257 that might impact GLS resistance by regulating gibberellin content, was further identified through haplotype-based association analysis, candidate gene expression analysis, and previous reports. CONCLUSIONS: The discovery of the novel candidate gene provides valuable genomic resources for elucidating the genetic mechanisms underlying GLS resistance in maize. Additionally, these findings will contribute to the development of new genetic resources by utilizing molecular markers to facilitate the genetic improvement and breeding of maize for GLS resistance.

ZmWAK02 encoding an RD-WAK protein confers maize resistance against gray leaf spot.

Gray leaf spot (GLS) caused by Cercospora zeina or C. zeae-maydis is a major maize disease throughout the world. Although more than 100 QTLs resistant against GLS have been identified, very few of them have been cloned. Here, we identified a major resistance QTL against GLS, qRglsSB, explaining 58.42% phenotypic variation in SB12×SA101 BC1 F1 population. By fine-mapping, it was narrowed down into a 928 kb region. By using transgenic lines, mutants and complementation lines, it was confirmed that the ZmWAK02 gene, encoding an RD wall-associated kinase, is the responsible gene in qRglsSB resistant against GLS. The introgression of the ZmWAK02 gene into hybrid lines significantly improves their grain yield in the presence of GLS pressure and does not reduce their grain yield in the absence of GLS. In summary, we cloned a gene, ZmWAK02, conferring large effect of GLS resistance and confirmed its great value in maize breeding.

Major biotic stresses affecting maize production in Kenya and their implications for food security.

Maize (Zea mays L.) is a staple food for many households in sub-Saharan Africa (SSA) and also contributes to the gross domestic product (GDP). However, the maize yields reported in most SSA countries are very low and this is mainly attributed to biotic and abiotic stresses. These stresses have been exacerbated by climate change which has led to long periods of drought or heavy flooding and the emergence of new biotic stresses. Few reports exist which compile the biotic stresses affecting maize production in SSA. Here, five major biotic stresses of maize in Kenya are presented which are attributed to high yield losses. They include Maize lethal necrosis, fall armyworm, gray leaf spot, turcicum leaf blight and desert locusts. Maize lethal necrosis and fall armyworm are new biotic stresses to the Kenyan maize farmer while gray leaf spot, and turcicum leaf blight are endemic to the region. The invasion by the desert locusts is speculated to be caused by climate change. The biotic stresses cause a reduction in maize yield of 30-100% threatening food security. Therefore, this review focuses on the cause, control measures employed to control these diseases and future prospective. There should be deliberate efforts from the government and researchers to control biotic stresses affecting maize yields as the effect of these stresses is being exacerbated by the changing climate.

Pyricularia's Capability of Infecting Different Grasses in Two Regions of Mexico.

The genus Pyricularia includes species that are phytopathogenic fungi, which infect different species of Poaceae, such as rice and sorghum. However, few isolates have been genetically characterized in North America. The current study addresses this lack of information by characterizing an additional 57 strains of three grasses (Stenotaphrum secundatum, Cenchrus ciliaris and Digitaria ciliaris) from two distant regions of Mexico. A Pyricularia dataset with ITS sequences retrieved from GenBank and the studied sequences were used to build a haplotype network that allowed us to identify a few redundant haplotypes highly related to P. oryzae species. An analysis considering only the Mexican sequences allowed us to identify non-redundant haplotypes in the isolates of C. ciliaris and D. ciliaris, with a high identity with P. pennisetigena. The Pot2-TIR genomic fingerprinting technique resulted in high variability and allowed for the isolates to be grouped according to their host grass, whilst the ERIC-PCR technique was able to separate the isolates according to their host grass and their region of collection. Representative isolates from different host grasses were chosen to explore the pathogenic potential of these isolates. The selected isolates showed a differential pathogenic profile. Cross-infection with representative isolates from S. secundatum and C. ciliaris showed that these were unable to infect D. ciliaris grass and that the DY1 isolate from D. ciliaris was only able to infect its host grass. The results support the identification of pathogenic strains of Pyricularia isolates and their cross-infection potential in different grasses surrounding important crops in Mexico.

Combination of linkage and association mapping with genomic prediction to infer QTL regions associated with gray leaf spot and northern corn leaf blight resistance in tropical maize.

Among the diseases threatening maize production in Africa are gray leaf spot (GLS) caused by Cercospora zeina and northern corn leaf blight (NCLB) caused by Exserohilum turcicum. The two pathogens, which have high genetic diversity, reduce the photosynthesizing ability of susceptible genotypes and, hence, reduce the grain yield. To identify population-based quantitative trait loci (QTLs) for GLS and NCLB resistance, a biparental population of 230 lines derived from the tropical maize parents CML511 and CML546 and an association mapping panel of 239 tropical and sub-tropical inbred lines were phenotyped across multi-environments in western Kenya. Based on 1,264 high-quality polymorphic single-nucleotide polymorphisms (SNPs) in the biparental population, we identified 10 and 18 QTLs, which explained 64.2% and 64.9% of the total phenotypic variance for GLS and NCLB resistance, respectively. A major QTL for GLS, qGLS1_186 accounted for 15.2% of the phenotypic variance, while qNCLB3_50 explained the most phenotypic variance at 8.8% for NCLB resistance. Association mapping with 230,743 markers revealed 11 and 16 SNPs significantly associated with GLS and NCLB resistance, respectively. Several of the SNPs detected in the association panel were co-localized with QTLs identified in the biparental population, suggesting some consistent genomic regions across genetic backgrounds. These would be more relevant to use in field breeding to improve resistance to both diseases. Genomic prediction models trained on the biparental population data yielded average prediction accuracies of 0.66-0.75 for the disease traits when validated in the same population. Applying these prediction models to the association panel produced accuracies of 0.49 and 0.75 for GLS and NCLB, respectively. This research conducted in maize fields relevant to farmers in western Kenya has combined linkage and association mapping to identify new QTLs and confirm previous QTLs for GLS and NCLB resistance. Overall, our findings imply that genetic gain can be improved in maize breeding for resistance to multiple diseases including GLS and NCLB by using genomic selection.

Population genomic analyses suggest recent dispersal events of the pathogen Cercospora zeina into East and Southern African maize cropping systems.

A serious factor hampering global maize production is gray leaf spot disease. Cercospora zeina is one of the causative pathogens, but population genomics analysis of C. zeina is lacking. We conducted whole-genome Illumina sequencing of a representative set of 30 C. zeina isolates from Kenya and Uganda (East Africa) and Zambia, Zimbabwe, and South Africa (Southern Africa). Selection of the diverse set was based on microsatellite data from a larger collection of the pathogen. Pangenome analysis of the C. zeina isolates was done by (1) de novo assembly of the reads with SPAdes, (2) annotation with BRAKER, and (3) protein clustering with OrthoFinder. A published long-read assembly of C. zeina (CMW25467) from Zambia was included and annotated using the same pipeline. This analysis revealed 790 non-shared accessory and 10,677 shared core orthogroups (genes) between the 31 isolates. Accessory gene content was largely shared between isolates from all countries, with a few genes unique to populations from Southern Africa (32) or East Africa (6). There was a significantly higher proportion of effector genes in the accessory secretome (44%) compared to the core secretome (24%). PCA, ADMIXTURE, and phylogenetic analysis using a neighbor-net network indicated a population structure with a geographical subdivision between the East African isolates and the Southern African isolates, although gene flow was also evident. The small pangenome and partial population differentiation indicated recent dispersal of C. zeina into Africa, possibly from 2 regional founder populations, followed by recurrent gene flow owing to widespread maize production across sub-Saharan Africa.

Pathogenicity Variation in Two Genomes of Cercospora Species Causing Gray Leaf Spot in Maize.

The gray leaf spots caused by Cercospora spp. severely affect the yield and quality of maize. However, the evolutionary relation and pathogenicity variation between species of the Cercospora genus is largely unknown. In this study, we constructed high-quality reference genomes by nanopore sequencing two Cercospora species, namely, C. zeae-maydis and C. zeina, with differing pathogenicity, collected from northeast (Liaoning [LN]) and southeast (Yunnan [YN]) China, respectively. The genome size of C. zeae-maydis-LN is 45.08 Mb, containing 10,839 annotated genes, whereas that of Cercospora zeina-YN is 42.18 Mb, containing 10,867 annotated genes, of which approximately 86.58% are common in the two species. The difference in their genome size is largely attributed to increased long terminal repeat retrotransposons of 3.8 Mb in total length in C. zeae-maydis-LN. There are 41 and 30 carbohydrate-binding gene subfamilies identified in C. zeae-maydis-LN and C. zeina-YN, respectively. A higher number of carbohydrate-binding families found in C. zeae-maydis-LN, and its unique CBM4, CBM37, and CBM66, in particular, may contribute to variation in pathogenicity between the two species, as the carbohydrate-binding genes are known to encode cell wall-degrading enzymes. Moreover, there are 114 and 107 effectors predicted, with 47 and 46 having unique potential pathogenicity in C. zeae-maydis-LN and C. zeina-YN, respectively. Of eight effectors randomly selected for pathogenic testing, five were found to inhibit cell apoptosis induced by Bcl-2-associated X. Taken together, our results provide genomic insights into variation in pathogenicity between C. zeae-maydis and C. zeina. [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.

Deep Learning Diagnostics of Gray Leaf Spot in Maize under Mixed Disease Field Conditions.

Maize yields worldwide are limited by foliar diseases that could be fungal, oomycete, bacterial, or viral in origin. Correct disease identification is critical for farmers to apply the correct control measures, such as fungicide sprays. Deep learning has the potential for automated disease classification from images of leaf symptoms. We aimed to develop a classifier to identify gray leaf spot (GLS) disease of maize in field images where mixed diseases were present (18,656 images after augmentation). In this study, we compare deep learning models trained on mixed disease field images with and without background subtraction. Performance was compared with models trained on PlantVillage images with single diseases and uniform backgrounds. First, we developed a modified VGG16 network referred to as "GLS_net" to perform binary classification of GLS, which achieved a 73.4% accuracy. Second, we used MaskRCNN to dynamically segment leaves from backgrounds in combination with GLS_net to identify GLS, resulting in a 72.6% accuracy. Models trained on PlantVillage images were 94.1% accurate at GLS classification with the PlantVillage testing set but performed poorly with the field image dataset (55.1% accuracy). In contrast, the GLS_net model was 78% accurate on the PlantVillage testing set. We conclude that deep learning models trained with realistic mixed disease field data obtain superior degrees of generalizability and external validity when compared to models trained using idealized datasets.

Transcriptomic Analysis Reveals Candidate Genes Responding Maize Gray Leaf Spot Caused by Cercospora zeina.

Gray leaf spot (GLS), caused by the fungal pathogen Cercospora zeina (C. zeina), is one of the most destructive soil-borne diseases in maize (Zea mays L.), and severely reduces maize production in Southwest China. However, the mechanism of resistance to GLS is not clear and few resistant alleles have been identified. Two maize inbred lines, which were shown to be resistant (R6) and susceptible (S8) to GLS, were injected by C. zeina spore suspensions. Transcriptome analysis was carried out with leaf tissue at 0, 6, 24, 144, and 240 h after inoculation. Compared with 0 h of inoculation, a total of 667 and 419 stable common differentially expressed genes (DEGs) were found in the resistant and susceptible lines across the four timepoints, respectively. The DEGs were usually enriched in 'response to stimulus' and 'response to stress' in GO term analysis, and 'plant-pathogen interaction', 'MAPK signaling pathways', and 'plant hormone signal transduction' pathways, which were related to maize's response to GLS, were enriched in KEGG analysis. Weighted-Genes Co-expression Network Analysis (WGCNA) identified two modules, while twenty hub genes identified from these indicated that plant hormone signaling, calcium signaling pathways, and transcription factors played a central role in GLS sensing and response. Combing DEGs and QTL mapping, five genes were identified as the consensus genes for the resistance of GLS. Two genes, were both putative Leucine-rich repeat protein kinase family proteins, specifically expressed in R6. In summary, our results can provide resources for gene mining and exploring the mechanism of resistance to GLS in maize.

Fine Mapping of a New Major QTL-qGLS8 for Gray Leaf Spot Resistance in Maize.

Gray leaf spot (GLS), caused by different species of Cercospora, is a fungal, non-soil-borne disease that causes serious reductions in maize yield worldwide. The identification of major quantitative trait loci (QTLs) for GLS resistance in maize is essential for developing marker-assisted selection strategies in maize breeding. Previous research found a significant difference (P < 0.01) in GLS resistance between T32 (highly resistant) and J51 (highly susceptible) genotypes of maize. Initial QTL analysis was conducted in an F2 : 3 population of 189 individuals utilizing genetic maps that were constructed using 181 simple sequence repeat (SSR) markers. One QTL (qGLS8) was detected, defined by the markers umc1130 and umc2354 in three environments. The qGLS8 QTL detected in the initial analysis was located in a 51.96-Mb genomic region of chromosome 8 and explained 7.89-14.71% of the phenotypic variation in GLS resistance in different environments. We also developed a near isogenic line (NIL) BC3F2 population with 1,468 individuals and a BC3F2-Micro population with 180 individuals for fine mapping. High-resolution genetic and physical maps were constructed using six newly developed SSRs. The QTL-qGLS8 was narrowed down to a 124-kb region flanked by the markers ym20 and ym51 and explained up to 17.46% of the phenotypic variation in GLS resistance. The QTL-qGLS8 contained seven candidate genes, such as an MYB-related transcription factor 24 and a C 3 H transcription factor 347), and long intergenic non-coding RNAs (lincRNAs). The present study aimed to provide a foundation for the identification of candidate genes for GLS resistance in maize.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 µm (average 8.24 µm) wide by 15.96 to 29.26 µm (average 25.40 µm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch's postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml-1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

High-density mapping for gray leaf spot resistance using two related tropical maize recombinant inbred line populations.

Gray leaf spot (GLS) caused by Cercospora zeae-maydis or Cercospora zeina is one of the devastating maize foliar diseases worldwide. Identification of GLS-resistant quantitative trait loci (QTL)/genes plays an urgent role in improving GLS resistance in maize breeding practice. Two groups of recombinant inbred line (RIL) populations derived from CML373 × Ye107 and Chang7-2 × Ye107 were generated and subjected to genotyping-by-sequencing (GBS). A total of 1,929,222,287 reads in CML373 × Ye107 (RIL-YCML) and 2,585,728,312 reads in Chang7-2 × Ye107 (RIL-YChang), with an average of 10,961,490 (RIL-YCML) and 13,609,096 (RIL-YChang) reads per individual, were got, which was roughly equal to 0.70-fold and 0.87-fold coverage of the maize B73 RefGen_V4 genome for each F7 individual, respectively. 6418 and 5139 SNP markers were extracted to construct two high-density genetic maps. Comparative analysis using these physically mapped marker loci demonstrated a satisfactory colinear relationship with the reference genome. 11 GLS-resistant QTL have been detected. The individual QTL accounted for 1.53-24.00% of the phenotypic variance explained (PVE). The new consensus QTL (qYCM-DS3-3/qYCM-LT3-1/qYCM-LT3-2) with the largest effect was located in chromosome bin 3.05, with an interval of 2.7 Mb, representing 13.08 to 24.00% of the PVE. Further gene annotation indicated that there were four candidate genes (GRMZM2G032384, GRMZM2G041415, GRMZM2G041544, and GRMZM2G035992) for qYCM-LT3-1, which may be related to GLS resistance. Combining RIL populations and GBS-based high-density genetic maps, a new larger effect QTL was delimited to a narrow genomic interval, which will provide a new resistance source for maize breeding programs.

Population genetic structure and migration patterns of the maize pathogenic fungus, Cercospora zeina in East and Southern Africa.

Cercospora zeina is a causal pathogen of gray leaf spot (GLS) disease of maize in Africa. This fungal pathogen exhibits a high genetic diversity in South Africa. However, little is known about the pathogen's population structure in the rest of Africa. In this study, we aimed to assess the diversity and gene flow of the pathogen between major maize producing countries in East and Southern Africa (Kenya, Uganda, Zambia, Zimbabwe, and South Africa). A total of 964 single-spore isolates were made from GLS lesions and confirmed as C.zeina using PCR diagnostics. The other causal agent of GLS, Cercospora zeae-maydis, was absent. Genotyping all the C.zeina isolates with 11 microsatellite markers and a mating-type gene diagnostic revealed (i) high genetic diversity with some population structure between the five African countries, (ii) cryptic sexual recombination, (iii) that South Africa and Kenya were the greatest donors of migrants, and (iv) that Zambia had a distinct population. We noted evidence of human-mediated long-distance dispersal, since four haplotypes from one South African site were also present at five sites in Kenya and Uganda. There was no evidence for a single-entry point of the pathogen into Africa. South Africa was the most probable origin of the populations in Kenya, Uganda, and Zimbabwe. Continuous annual maize production in the tropics (Kenya and Uganda) did not result in greater genetic diversity than a single maize season (Southern Africa). Our results will underpin future management of GLS in Africa through effective monitoring of virulent C.zeina strains.

Quantitative Trait Loci Associated with Gray Leaf Spot Resistance in St. Augustinegrass.

Gray leaf spot (GLS), caused by Magnaporthe grisea, is a major fungal disease of St. Augustinegrass (Stenotaphrum secundatum), causing widespread blighting of the foliage under warm, humid conditions. To identify quantitative trait loci (QTL) controlling GLS resistance, an F1 mapping population consisting of 153 hybrids was developed from crosses between cultivar Raleigh (susceptible parent) and plant introduction PI 410353 (resistant parent). Single-nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing constituted nine linkage groups for each parental linkage map. The Raleigh map consisted of 2,257 SNP markers and spanned 916.63 centimorgans (cM), while the PI 410353 map comprised 511 SNP markers and covered 804.27 cM. GLS resistance was evaluated under controlled environmental conditions with measurements of final disease incidence and lesion length. Additionally, two derived traits, area under the disease progress curve and area under the lesion expansion curve, were calculated for QTL analysis. Twenty QTL were identified as being associated with these GLS resistance traits, which explained 7.6 to 37.2% of the total phenotypic variation. Three potential GLS QTL "hotspots" were identified on two linkage groups: P2 (106.26 to 110.36 cM and 113.15 to 116.67 cM) and P5 (17.74 to 19.28 cM). The two major effect QTL glsp2.3 and glsp5.2 together reduced 20.2% of disease incidence in this study. Sequence analysis showed that two candidate genes encoding ß-1,3-glucanases were found in the intervals of two QTL, which might function in GLS resistance response. These QTL and linked markers can be potentially used to assist the transfer of GLS resistance genes to elite St. Augustinegrass breeding lines.

De novo Assembly of Transcriptomes From a B73 Maize Line Introgressed With a QTL for Resistance to Gray Leaf Spot Disease Reveals a Candidate Allele of a Lectin Receptor-Like Kinase.

Gray leaf spot (GLS) disease in maize, caused by the fungus Cercospora zeina, is a threat to maize production globally. Understanding the molecular basis for quantitative resistance to GLS is therefore important for food security. We developed a de novo assembly pipeline to identify candidate maize resistance genes. Near-isogenic maize lines with and without a QTL for GLS resistance on chromosome 10 from inbred CML444 were produced in the inbred B73 background. The B73-QTL line showed a 20% reduction in GLS disease symptoms compared to B73 in the field (p = 0.01). B73-QTL leaf samples from this field experiment conducted under GLS disease pressure were RNA sequenced. The reads that did not map to the B73 or C. zeina genomes were expected to contain novel defense genes and were de novo assembled. A total of 141 protein-coding sequences with B73-like or plant annotations were identified from the B73-QTL plants exposed to C. zeina. To determine whether candidate gene expression was induced by C. zeina, the RNAseq reads from C. zeina-challenged and control leaves were mapped to a master assembly of all of the B73-QTL reads, and differential gene expression analysis was conducted. Combining results from both bioinformatics approaches led to the identification of a likely candidate gene, which was a novel allele of a lectin receptor-like kinase named L-RLK-CML that (i) was induced by C. zeina, (ii) was positioned in the QTL region, and (iii) had functional domains for pathogen perception and defense signal transduction. The 817AA L-RLK-CML protein had 53 amino acid differences from its 818AA counterpart in B73. A second "B73-like" allele of L-RLK was expressed at a low level in B73-QTL. Gene copy-specific RT-qPCR confirmed that the l-rlk-cml transcript was the major product induced four-fold by C. zeina. Several other expressed defense-related candidates were identified, including a wall-associated kinase, two glutathione s-transferases, a chitinase, a glucan beta-glucosidase, a plasmodesmata callose-binding protein, several other receptor-like kinases, and components of calcium signaling, vesicular trafficking, and ethylene biosynthesis. This work presents a bioinformatics protocol for gene discovery from de novo assembled transcriptomes and identifies candidate quantitative resistance genes.