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The sorghum association panel was evaluated for grain mold severity, seed weight, and germination rate. The 377 accessions were inoculated with Alternaria alternata alone, a mixture of A. alternata, Fusarium thapsinum, and Curvularia lunata, and untreated water-sprayed control during 2010, 2013-2015 growing seasons at the Texas AgriLife Research Farm, Burleson County, Texas. Each accession was evaluated at least twice. Across accessions, Spearman’s rank correlation was performed for non-parametric correlation analysis for grain mold severity, seed weight, and germination rate. There were significant negative correlations between grain mold severity with seed weight and germination rate for the individual treatment and when combined. A significant positive correlation between seed weight and germination rate was observed. The results indicated that higher grain mold severity reduces both sorghum seed weight and germination rate whether deliberately inoculated with fungal pathogens or naturally infected. It can be argued that correlations from this study were more robust due to a large number of accessions from all major sorghum races used and may represent the true association among the three parameters for this pathosystem. Thus, the use of grain mold-resistant lines, resulting in sound seeds and higher germination rates is recommended.

Johnsongrass [Sorghum halepense (L.) Pers.] is one of the most noxious weeds distributed around the world. Due to its rapid growth, wide dissemination, seeds that can germinate after years in the soil, and ability to spread via rhizomes, S. halepense is difficult to control. From a perspective of plant pathology, S. halepense is also a potential reservoir of pathogens that can eventually jump to other crops, especially corn (Zea mays L.) and sorghum [Sorghum bicolor (L.) Moench]. As one of the most problematic weeds, S. halepense and its diseases can provide useful information concerning its role in diseases of agronomically important crops. An alternative consideration is that S. halepense may provide a source of genes for resistance to pathogens. While some studies have verified that pathogens isolated from S. halepense actually cause disease on host crops through cross inoculation, similarity of disease symptoms and pathogen morphology have been used for identity of the disease agent in most studies. Availability of DNA sequence information has greatly altered and improved pathogen identification, leading to significant changes in phylogenetic assignments. Reclassification of pathogens, especially fungi, raises new questions concerning the role of S. halepense as a disease reservoir. Our goals in this review are to pinpoint, where possible, diseases for which S. halepense acts as a significant pathogen reservoir and to point out problem areas where further research is needed.

Cotton production in Xinjiang, the largest cotton-producing area in China, has an increasingly serious disease threat from Verticillium dahliae. Eighty-five V. dahliae isolates were obtained from wilted cotton plants collected from eight counties in Xinjiang. The isolates were assessed for genotypic diversity by DNA sequence analysis and PCR molecular genotyping with specific markers for race 1, race 2, defoliating (D) pathotype, nondefoliating (ND) pathotype, and mating type idiomorph Mat1-2. Isolates belonged to lineages 1A or 2B, with three subgenotypes found in each lineage. All isolates tested positive for race 2 and Mat1-2 markers. All isolates in lineage 2B tested positive for the ND pathotype marker but only isolates in the major subgenotype in lineage 1A tested positive for the D pathotype marker. Pathogenicity assays on Gossypium hirsutum ‘Acala 44’ demonstrated no significant difference among subgenotypes within each lineage. Isolates in lineage 1A caused greater shoot weight reductions, percent leaf drop, and percent diseased leaves than isolates in lineage 2B. One isolate in each lineage for 1A and 2B was avirulent. Isolates in lineage 1A caused greater than 50% leaf drop and a 17-g shoot weight reduction compared with a 9% leaf drop and a 6-g shoot weight reduction by isolates in lineage 2B. Overall, 42% of the V. dahliae isolates from Xinjiang were D pathotype but the percentage varied widely among locations. Two plants had both pathotypes. Nineteen isolates of Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) also were recovered from wilted plants in Xinjiang. Two plants had both Verticillium wilt and Fusarium wilt pathogens. Both pathogens should be considered when using or developing wilt resistant or tolerant materials for Xinjiang.

This study was conducted to identify resistance sources against the newly documented pathotypes (P5 and P6) of Sporisorium reilianum, causing sorghum head smut. A subset of 67 sorghum association panel (SAP) accessions, 29 in 2017 and 38 in 2018 along with checks BTx635 (resistant) and BTx643 (susceptible) were screened in the greenhouse against P5 and P6 pathotypes in two separate experiments in both years. At 18 to 20 days after planting, accessions were inoculated by injecting the seedlings below the apical meristem with sporidial suspensions following an established inoculation procedure. Three accessions (PI656091, PI533919, and PI533821) in 2017 and 17 accessions (PI597961, PI656071, PI656048, PI576435, PI534075, PI534145, PI656057, PI576437, PI651492, PI656014, PI656025, PI655999, PI656010, PI656082, PI534123, PI533927, PI656100) in 2018 were identified as potential resistant sources to both P5 and P6 pathotypes of S. reilianum. These SAP accessions are new sources that can be further used to develop new breeding parental lines and hybrids and mapping populations for quantitative trait loci markers closely linked to head smut resistance.

Basal leaf angle distribution was surveyed in twenty-one Johnsongrass cultivars near the end of the vegetative stage. The angles increased from the top to the bottom leaves, and compared to cultivated grain sorghums, the average angle was larger in Johnsongrass. When basal leaf angle distribution data were correlated with pathogenicity test data from excised-leaf assays for three isolates of Colletotrichum sublineola, the results showed a weak positive correlation between basal leaf angle and pathogenicity level in Johnsongrass. In order to investigate a protective role of leaf thickness to C. sublineola, leaf thickness was measured in three sorghum cultivars and one Johnsongrass cultivar at the 8-leaf-stage. Leaf thickness near the apex, near the base, and half-way between the two points were measured in the top four leaves of each plant. Thickness of leaf blade and midrib were recorded separately. Using an excised-leaf-assay, the three points were inoculated with C. sublineola, and pathogenicity level was recorded 4-days-post-inoculation. Results showed strong negative correlations between leaf midrib thickness and pathogenicity level in sorghum and Johnsongrass but not in leaf blades.

A total of 377 lines from Sorghum Association Panel (SAP) grouped into 10 subpopulations based on eight phenotypic traits, including flag leaf width, terminal branch length, flag leaf height, and flowering time and genetic data were evaluated for grain mold resistance by inoculating them with Alternaria alternata alone, a mixture of A. alternata, Fusarium thapsinum, and Curvularia lunata, and untreated water-sprayed control during 2010, 2013–2015 growing seasons at the Texas A&M AgriLife Research Farm, Burleson County, Texas. Each accession was evaluated at least twice. Eleven accessions exhibited grain mold severity ratings of 2 or less, indicating that they were resistant to highly resistant to the disease when challenged with A. alternata alone, 7 lines identified as such when inoculated with a mixture of A. alternata, F. thapsinum, and C. lunata, and 17 resistant lines were identified under the untreated control. Three lines PI533871, PI576130, and PI656036 exhibited resistant to highly resistant response to grain mold across treatments. When the 10 subpopulations were compared for disease response, accessions within subpopulation 4 had the lowest overall grain mold severity ratings, indicating that this subpopulation (zerazera/caudatum racial type) harbors genes that may contribute to grain mold resistance. A genome-wide association study with over 79,000 single-nucleotide polymorphic (SNP) loci from a publicly available genotype by sequencing dataset available for the (SAP) lines was conducted to identify genomic regions associated with grain mold resistance response based on the individual and combined treatments. In most cases, the top-scoring SNPs were mapped to the nearest or a nearby annotated gene with precedence for a role in host defense.

Colletotrichum sublineola is a casual pathogen of sorghum anthracnose. Sorghum pathologists often need to conduct evaluations for anthracnose resistance in large scale which are expensive and labor intensive. As a solution, an excised-leaf assay has been used, but whether or not leaf age, position or region affects pathogenicity scores has not previously been evaluated. Essentially, in an excised-leaf assay, is response to C. sublineola over all or part of a leaf blade identical? To get an answer, three sorghum and one Johnson grass cultivars were tested. The top five leaves were inoculated at the apex, mid-leaf and base of each leaf blade. Results show nearly no effect of leaf age to pathogenicity level within the top five leaves. Furthermore, in order to evaluate any protective role of leaf wax to C. sublineola, the wax was disrupted by simply wiping the leaf surface by a thumb, or as an alternative method, leaf surface tension was reduced by submerging leaves into 2% TWEEN 20 before inoculation. Compared to control, wiped leaves increased pathogenicity scores on the leaf blade and midrib in two of three sorghum cultivars, but 2% TWEEN 20-treated leaves had only minimal changes in pathogenicity level compared to controls.

Aims: The aim of this study was to identify resistant accessions against pathogens, causing anthracnose and grain mold. Study Design: Study was laid out in a randomized complete block design with three replications. Data for anthracnose rating, grain mold severity, seed weight, and percent germination rate were analyzed using the command PROC GLM. Place and Duration of Study: The study was carried out at the Texas AgriLife Research Farm, Burleson County, Texas, in 2010, 2014, and 2015 growing seasons. Methodology: Forty-seven accessions were planted in 6 m rows 0.31 m spacing. Plants were inoculated by placing Colletotrichum sublineola colonized grain in the plant whorls 30 days after planting. Disease evaluation was initiated 30 days post-inoculation and thereafter on a weekly basis for three consecutive weeks. Grain mold experiment: Three treatments were used: 1) plants sprayed with A. alternata alone, 2) a mixture of A. alternata, F. thapsinum and C. lunata, 3) control plants sprayed with sterilized water and exposed to natural infection. At 50% bloom, three panicles per line within replication were inoculated for each treatment. Results: Eleven accessions, including PI641874, PI656070, PI656115, and PI534167 were consistently resistant when challenged with the anthracnose pathogen, C. sublineola. Accessions PI534047 and PI574455, exhibited resistance to moderately resistance grain mold response when challenged with the treatments. Seed weight, germination rate, and mycoflora analysis which are factors in determining grain mold resistance also were measured. Across the accessions, mean seed weight ranged from1.4 g to 4.3 g per 100 kernel and germination rate ranged from 26 to 87%. Conclusion: The resistant accessions identified in this study can be used in breeding programs to develop anthracnose and grain mold resistance lines.

Cotton plants produce two classes of terpenoid defence compounds against pathogens and other pests. Both classes are derived from a common sesquiterpenoid precursor, δ‐cadinen‐2‐one, which enters either the gossypol pathway or the lacinilene pathway. Blocking the gossypol pathway by RNAi suppression of the early pathway biosynthetic enzyme CYP82D hydroxylase resulted in enhanced resistance to the Fusarium wilt pathogen. Analyses of root terpenoids revealed no overall increases in the products of the gossypol pathway in the roots infected by the wilt pathogen. However, the lacinilene pathway was elicited by the pathogen and the lacinilene levels were 19‐fold higher in the RNAi plants than in wild‐type plants. In the pathogen inoculated RNAi 73R plants, the concentrations of DHC and HMC were 231 μg and 886 μg/g dry roots, respectively, which may have contributed to the inhibition of fungal invasion. In comparison, the concentrations of DHC and HMC in the pathogen inoculated control wild‐type 73W plants were only 0.7 μg and 58 μg/g dry roots, respectively. Fungitoxicity testing showed that DHC at 100 μg/ml inhibited growth of the Fusarium wilt pathogen by >93%. Treatment with the phytohormone jasmonic acid failed to elicit production of lacinilene pathway terpenoids in roots of either RNAi plants or their wild‐type sibling lines, but increased production of gossypol pathway terpenoids with concentrations in RNAi plants 80%–97% less than those in wild‐type plants. This indicates that induction of the lacinilene pathway is not directly mediated by jasmonic acid signalling and requires other signalling to activate the pathway. These results illustrate possible mechanisms of wilt disease resistance in cotton and provide a new approach to increase host resistance by manipulating these two major cotton chemical defence pathways.

Three isolates of Colletotrichum sublineolum P. Henn were inoculated on leaf blades and midribs of twenty-six johnsongrass cultivars with two sorghum cultivars, BTx 623 and SC 748-5 by using an excised leaf method. Phenotypic host responses from no response to severe infection, were observed. Some johnsongrass cultivars and BTx 623 showed different responses between the two tissues. Expression of six defense response related genes were measured using Real-time qRT-PCR. Leaf blade and midrib showed different patterns in BTx 623 inoculated with C. sublineolum; high expression of CHS8 and TLP were measured in midrib at 24 and 48 hpi respectively.

Gene expression was analyzed at 0- and 24-h post-inoculation of two inbred sorghum cultivars known to differ in response to inoculation with Colletotrichum sublineola, the fungal pathogen that causes anthracnose. QL3 is reported to have quantitative resistance, while Theis is susceptible to most pathotypes of the pathogen; RNASeq identified over 3,000 specific genes in both cultivars as showing significant changes in expression following inoculation; in all but one gene, the changes in QL3 and Thies were in the same direction. Many other genes showed significant changes in only one of the two cultivars. Overall, more genes were downregulated than upregulated. Differences in changes in expression levels of a few genes suggested potential roles for the difference in disease response between QL3 and Theis, but did not identify known resistance genes. Gene ontology (GO) and pathway enrichment analysis identified upregulation of 23 transcription factor encoding genes as well as genes involved in the production of secondary metabolites, which are part of a typical host defense reaction.

Anthracnose disease of sorghum is caused by Colletotrichum sublineola, a filamentous fungus. The genetic basis of resistance to anthracnose in sorghum is largely unclear, especially in Senegalese sorghum germplasm. In this study, 163 Senegalese sorghum accessions were evaluated for response to C. sublineola, and a genome‐wide association study (GWAS) was performed to identify genetic variation associated with response to C. sublineola using 193,727 single nucleotide polymorphisms (SNPs) throughout the genome. Germplasm diversity analysis showed low genetic diversity and slow linkage disequilibrium (LD) decay among the Senegalese accessions. Phenotypic analysis resulted in relatively low differences to C. sublineola among the tested population. Genome‐wide association study did not identify any significant association based on a strict threshold for the number of SNPs available. However, individual analysis of the top eight SNPs associated with relative susceptibility and resistance identified candidate genes that have been shown to play important roles in plant stress tolerance in previous studies. This study identifies sorghum genes whose annotated properties have known roles in host defense and thus identify them as candidates for use in breeding for resistance to anthracnose.

Sporisorium reilianum causes head smut in sorghum and maize and exists in two formae speciales, Sporisorium reilianum f. sp. reilianum and Sporisorium reilianum f. sp. zeae that have preference for sorghum and maize, respectively. Infection requires the formation of a dikaryon between sporidia of compatible mating types, leading to a change from yeast-like to hyphal growth within the host plant. This switching is controlled through mating type loci. A total of 66 sorghum isolates of S. reilianum collected from fields in different geographic regions and 2 maize isolates were examined for mating compatibility, leading to the establishment of haploid cultures with three different alleles at the a mating type locus, as verified by gene expression. Interestingly, a mating compatibility was detected between maize and sorghum isolates. Comparison of amino acid sequences, deduced from nucleotide sequencing of pheromone precursor genes of sorghum to the corresponding components in maize, showed 100% similarity of pheromone components mfa1.2, mfa2.1, mfa3.1 and 97% for mfa1.3, mfa3.2 and mfa2.3. Only 1 amino acid substitution was detected in sorghum mfa1.3. To assay host preference in relationship to whole genome polymorphism, six amplified fragment length polymorphism (AFLP) selective primer combinations were used on DNA of S. reilianum isolates. High genetic polymorphism (61%) was observed between sorghum and maize isolates. The resultant dendrogram constructed using neighbor-joining (NJ) analysis grouped maize isolates into one cluster with high similarity (>88%) while sorghum isolates grouped into four clusters suggesting that genetic differentiation contributes to host specific populations of S. reilianum.

Twenty-six johnsongrass cultivars, collected from seven U.S. states, were spray inoculated with spores of an isolate of Colletotrichum sublineola derived from sorghum. Inoculations were made when plants reached growth stages of 2 (four to five leaves), 3 (seven to eight leaves), and 6 (post panicle emergence). At the earlier stages the johnsongrass cultivars showed hypersensitive responses and small lesions; however, acervulus formation was not observed. When late growth stage johnsongrass plants were spray inoculated, acervulus formation was confirmed in 19 cultivars as early as 5 days postinoculation. Results obtained confirm that johnsongrass can act as an alternate host of C. sublineola under favorable conditions, and growth stages of johnsongrass may be an important factor for cross infection of C. sublineola between johnsongrass and sorghum.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A highly virulent race 4 (Cal race 4) of Fusarium oxysporum f. sp. vasinfectum was identified in California cotton fields in 2001, and has since been found in increasing numbers of fields. Cal race 4 isolates contain a unique Tfo1 transposon insertion in the PHO gene that was not found in other F. oxysporum f. sp. vasinfectum genotypes. Based on this insertion, a multiplex polymerase chain reaction method was developed to detect the Cal race 4 pathogen. A panel of F. oxysporum f. sp. vasinfectum isolates representing different vegetative compatibility groups (VCG) and DNA sequence types was assembled to test the specificity of the detection method. In all, 16 of 17 Cal race 4 isolates produced a 583-bp amplicon; the other isolate produced a 396-bp amplicon reflecting the absence of the Tfo1 insertion. This isolate was a moderately virulent pathogen among Cal race 4 isolates. In total, 80 other F. oxysporum isolates associated with cotton and 11 other formae speciales of F. oxysporum produced only the 396-bp amplicon. The method also distinguished Cal race 4 isolates from India race 4 isolates and China race 7 isolates, which did not possess the unique Tfo1 insertion but otherwise had identical DNA sequences, and all belong to VCG0114. The method is capable of detecting the pathogen directly from infected stem tissues even before external symptom appears and, thus, provides an effective tool for timely identification of infested fields and seed lots, and should help reduce dissemination of Cal race 4 in the U.S. Cotton Belt.

SNPs identify prospective genes related to response to Colletotrichum sublineola (anthracnose) in the sorghum association panel lines. Sorghum association panel (SAP) lines were scored over several years for response to Colletotrichum sublineola, the causal agent of the disease anthracnose. Known resistant and susceptible lines were included each year to verify successful inoculation. Over 79,000 single-nucleotide polymorphic (SNP) loci from a publicly available genotype by sequencing dataset available for the SAP lines were used with TASSEL association mapping software to identify chromosomal locations associated with differences in disease response. When the top-scoring SNPs were mapped to the published sorghum genome, in each case, the nearest annotated gene has precedence for a role in host defense.

Abundant, frequent rains along with humid and cloudy conditions during the early part of the 2015 growing season provided conducive conditions for an unusually severe outbreak of zonate leaf spot and rough leaf spot in a block of sorghum lines at the Texas A&M AgriLife Research Farm, Burleson County, near College Station, Texas. This afforded the opportunity to evaluate these lines for reaction to these diseases, which normally occur at trace levels in this area. Out of 181 sorghum lines, including a subset of the sorghum association panel, a total of 13 lines, including Dorado, Sureno, PI576434, PI656005, PI656034, PI656075, PI656024, and PI598070, were resistant to both diseases. These lines could be used in breeding programs to introgress the genes for resistance to both diseases into other adapted lines. Accepted for publication 8 December 2015. Published 14 December 2015.

The recent outbreak of sorghum downy mildew (SDM) in Texas, USA caused by pathotype P3 of Peronosclerospora sorghi, which is resistant to standard fungicide seed treatment, and the subsequent development of a new pathotype, P6, that overcame resistance in some hybrids, has emphasized the importance of continuing to develop new sources with genetic resistance. Eighty-two exotic Gambian and Malian germplasms and 10 sorghum lines commonly used as SDM pathotype differentials were field-evaluated in a randomized complete block design replicated three times at one Mexican location (Ocotlan, Jalisco) in 2004 and 2005, and two USA locations (Louise and New Taiton, TX, USA) in 2005 to identify new sources of SDM resistance. Accessions PI609151 and PI609442 from Mali had high levels of SDM resistance at all locations. Malian accession PI612815 also had a moderate to resistant reaction to SDM in two of the three locations. Accession PI522108 from Gambia was resistant in Mexico but susceptible in Louise, TX, USA. The reaction of the 10 lines used as differentials suggested the presence of a pathotype in Mexico that differed from those in the USA.

In previous studies, a sorghum mini core collection was scored over several years for response to Colletotrichum sublineola, Peronosclerospora sorghi, and Sporisorium reilianum, the causal agents of the disease anthracnose, downy mildew, and head smut, respectively. The screening results were combined with over 290,000 Single nucleotide polymorphic (SNP) loci from an updated version of a publicly available genotype by sequencing (GBS) dataset available for the mini core collection. GAPIT (Genome Association and Prediction Integrated Tool) R package was used to identify chromosomal locations that differ in disease response. When the top scoring SNPs were mapped to the most recent version of the published sorghum genome, in each case, a nearby and most often the closest annotated gene has precedence for a role in host defense.

Amplified fragment length polymorphism (AFLP) based genetic diversity was analyzed for 232 Colletotrichum sublineolum isolates collected between 2002 and 2004 from three geographically distinct regions of Texas, and from Arkansas, Georgia, and Puerto Rico. Results revealed significant levels of polymorphism (59%) among the isolates. Even so, genetic similarity between isolates was high, ranging from 0.78 to 1.00. Clustering of similar isolates did not correlate with either geographic origin or year of collection. Pathotypes of 20 of the isolates were determined using 14 sorghum lines previously used in Brazil and the United States and 4 from Sudan. Seventeen new pathotypes were established from the 18 isolates that gave uniform and consistent reactions on all host differentials over 2 years of greenhouse testing. Differentials BTx378 and QL3 were resistant to all isolates while BTx623 and TAM428 were universally susceptible both years. Each of these lines had shown differential responses in prior studies indicating that the pathogen population has sufficient diversity to adapt rapidly to changes in resistant host lines deployed. When the 2-step pathotype classification scheme was used, the 18 isolates examined in this study were placed in four pathotype groups (A, C, D and G), which would further then be separated into ten distinct pathotypes. Common sets of differentials and a standardized nomenclature will allow for comparison to be made among pathotypes of C. sublineolum detected from different regions and also could help direct planting of appropriate sorghum lines and aid in the development of more durable forms of resistance.

Sorghum accessions from Uganda were evaluated for grain mold and anthracnose resistance during the 2005 and 2006 growing seasons at the Texas A&M University Research Farm, near College Station, TX. Accession PI534117 and SC719-11E exhibited the lowest grain mold severities of 2.4, whereas, accessions PI534117, PI534144, PI576337, PI297199, PI533833, and PI297210, with SC748-5 were highly resistant to anthracnose in both years. Accessions PI534117, PI297134, PI297156 exhibited low grain mold severities in 2006. Significant negative correlation was recorded between grain mold and percent germination and high temperature in both years. In 2006, daily precipitation recorded significant positive correlation with grain mold. The seed mycoflora was analyzed across sorghum lines and treatments. In 2005, Curvularia lunata and Fusarium thapsinum were the most frequently recovered fungal species with 31 and 21% incidence, respectively, followed by Alternaria spp. (19%) and F. semitectum (13%). In 2006, predominant colonizers were F. thapsinum (58%), followed by Alternaria and F. semitectum with 15 and 10, percent respectively, while C. lunata had a 6% incidence. In this study, PI534117 holds promise for multiple disease resistance, as it had the lowest disease severity of grain mold and was highly resistant to anthracnose in both years. It also has a high germination rate, a high seed weight, and its short stature is more advantageous for the new A-line conversion program.

Head smut, caused by the fungal pathogen Sporisorium reilianum, has been reported with increasing frequency in the grain sorghum growing areas of Texas. To facilitate analysis of changes in pathogen virulence, four inoculation techniques were examined: soil and teliospore mixture, seed coating, media placement, and syringe injection. Of the four, syringe injection was determined to be the most effective. Inoculations of sorghum host differentials BTx643, BTx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), and Tx414 showed 23 of 32 Texas isolates were race 4. Two isolates from College Station, TX, were classified as race 1, but no race 2 or 3 isolates were found. New, virulent races 5 and 6 were identified among isolates from south Texas. Using 16 amplified fragment length polymorphism (AFLP) primer combinations, genetic diversity was assessed in DNA samples from 49 S. reilianum isolates, including 44 sorghum isolates from Texas, two from Uganda, and one from Mali; and two maize isolates from Mexico. Single-base extensions with EcoRI and MseI primers in the selective amplification increased the number of informative polymorphic bands. High genetic dissimilarity (50%) was observed between isolates originating from maize and those originating from sorghum. The resultant dendrogram, made using cluster analysis, grouped the Texas S. reilianum isolates into four small clusters with ≥82% similarity. Other than for two race 6 isolates from Weslaco, TX, no evidence for geographical or other restrictions on gene flow was evident.

Globally, the foliar phase of anthracnose is one of the most destructive diseases of sorghum. In most cases, anthracnose resistance screening relies on the use of a spore suspension. This method is usually conducted after sundown and when there is the possibility of dew formation the following morning. Using a spore suspension for sorghum anthracnose field evaluation in College Station, Texas over five years (1996, 1997, 1999-2001) yielded inconsistent linkage results and failed to identify any closely linked molecular markers. For large scale screening of sorghum germplasm for anthracnose (Colletotrichum sublineolum) resistance, plants are inoculated in the field or in the green house at either 30 d after planting or at the 8-10 leaf-stage. In field inoculation, the use of C. sublineolum-colonized sorghum grains was shown to be the most efficient and effective in identifying resistant sources. For effective, efficient, fast and accurate infection, approximately 10-20 seeds are placed in each plant leaf whorl and it takes about 16.7 kg of colonized grains to cover a 0.4 ha area. In the greenhouse, though colonized grains are equally effective, spray inoculation is preferred for easy and uniform coverage. Using this method of inoculum preparation, spore suspension was extracted and sprayed (106 conidia·ml-1), followed by 10 hr/d misting for 30 sec at 30-45 min interval continuously for a period of one month resulted in effective infection.

Forty-six converted exotic sorghum lines representing all fi ve races and nine intermediate races of sorghum were fi ngerprinted using amplifi ed fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. A total of 453 scored AFLP and SSR loci were used to calculate genetic similarities between the lines. The dendrogram constructed using UPGMA grouped 31 lines into three major clusters with Jaccard coeffi cients greater than 0.75. The remaining 15 lines were grouped into four small sub-clusters each with two lines and seven single accession nodes. Phenetic analysis using AFLP and SSR markers resulted in clusters corresponding to the kafi r, guinea, caudatum and durra morphological groupings. Cluster and principal coordinate analyses indicate that the guinea, kafi r and intermediate lines in this study are more closely related than phenotype would suggest. Likewise, caudatum and intermediates involving caudatum showed close genetic relationship with durra and durra intermediates. For the most part, morphological classifi cation of race based on panicle traits was also refl ected by similarity in DNA based polymorphisms. The molecular diversity of bicolor and associated intermediate races is not refl ective of their common morphological classifi cation, since this race and its intermediates are quite heterogenous.

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F1 and F2 plants were inoculated with the local isolates of C. graminicola cultures. The F2 plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 011437 was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 011437 was cloned and sequenced. The sequence of RAPD marker OPJ 011437 was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 011437 and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 011437 was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.

Cotton quality and yield are affected by several factors during the growing season. A soil inhabiting fungus, Verticillium dahliae Kleb., can cause substantial yield loss in cotton. A molecular mapping F2 population derived from the interspecific cross of the highly tolerant Gossypium barbadense cv. Pima S-7 and the susceptible G. hirsutum cv. Acala 44 was phenotyped for disease incidence and severity. Phenotyping of individual plant reactions to the disease was quantified using a set of growth parameters measured 3 weeks after inoculation. The F2 phenotypic distribution of these parameters (number of healthy leaves, node number, leaf weight, stem weight, and total shoot weight) suggested that resistance is polygenic inherited. Microsatellites were used to reveal polymorphism between resistant and susceptible parents. A total of 255 simple sequence repeat (SSR) primer pairs were screened over bulks constituted by 10 resistant and 10 susceptible progeny. Sixty markers were used to analyze quantitative trait loci (QTLs). Eleven linkage groups were constructed consisting of 35 markers and spanning 531 cM with an average distance of 15.17 cM. QTL analysis was performed with MapQTL and QTL Cartographer. MapQTL indicated that 15 markers have significant linkage associations and 9 were distributed to chromosomes 10, 11, 12, and 25. Interval mapping also indicated the most likely position of markers that are significant and located on linkage groups. Three loci (CM12, STS1, 3147-2) had large effect on resistance to Verticillium wilt. Two loci were located on LG-1 and one on LG-2 and both linkage groups are located on chromosome 11. # 2005 Elsevier Ireland Ltd. All rights reserved.

Four Verticillium dahliae isolates (V76, TS-2, PH, and V44) were used in screening four cotton cultivars (Pima S-7, Acala Prema, M-315 and Acala 44). Pima S-7 and Acala Prema gave the highest resistance reactions and Acala 44 was the most susceptible. Isolate V76 of V. dahliae was the most virulent. An interspecific cross between the resistant cv Pima S-7 (Gossypium barbadense) and the susceptible cv. Acala 44 (G. hirsutum) was made and the F 2 population pheno-typed for Verticillium wilt effect. Phenotyping of plant reaction to the disease was quantified by using a set of six growth parameters (number of healthy leaves, number of nodes, leaf weight, stem weight, leaf to stem ratio, and total shoot weight) measured 3 weeks after inoculation. The F 2 phenotypic distribution of these parameters suggests that distribution is towards resistance and polygenic. Transgressive segregation also was observed. The number of healthy leaves and total shoot weight were found to be the best indicators of resistance. Results obtained in this study will be useful to quantify resistance to V. dahliae and identify the best parameters to phenotype in genetic studies.

A new cotton variant with reduced levels of terpenoid aldehydes (sesquiterpenoids and sesterterpenoids (heliocides)) was isolated from the progeny of hemizygous cotton ( Gossypium hirsutum cv. Coker 312) transformed with antisense (+)-δ-cadinene synthase cDNA. Southern analysis of leaf DNA digested with Hin dIII, Pst or Kpn I restriction endonucleases did not detect any antisense cdn1-C1 DNA in the genome of the variant. The gossypol content in the seed of the variant was markedly lower than in the seed of T1 antisense plants. Eighty-nine percent of the variant seed had a 71.1% reduction in gossypol and the foliage of the variant plants showed a 70% reduction in gossypol and a 31% reduction in heliocides. Compared to non-transformed plants there was no reduction in the number of lysigenous glands in the seed of the variant. The cotton variant shows uncoupling of terpenoid aldehyde synthesis and gland formation. The cotton variant may have resulted from somaclonal variation occurring in the callus tissue during the transformation–regeneration process.

Grain mold of Sorghum bicolor is one of the leading constraints for the production of optimum quality sorghum grain worldwide. Differences in mold levels among different varieties grown in the same environment imply that genes play a role in controlling mold severity. In order to determine if genes that function in active defense responses also affect molding, panicles of resistant and susceptible cultivars were inoculated at anthesis with conidial suspensions of Fusarium thapsinum and Curvularia lunata, the fungi most often found in naturally infected grain. RNA was extracted from the immature floral tissues at various times following inoculation. Levels of mRNA for four known defense-response genes, phenylalanine ammonia lyase (PAL1-1), chalcone synthase (CHS2G), β-1,3-glucanase (GLUC2-1) and chitinase (CHIT25-1) were examined by hybridization to PCR generated clones of the respective genes. Expression of each gene increased rapidly following inoculation with either fungus. Although differences were seen in response to the two pathogens, the general pattern was similar in resistant and susceptible cultivars. The results imply that factors other than the level or timing of active defense responses account for the cultivar differences seen when the plants are challenged at the time of flowering.

Induction of genes needed to synthesize terpenoid compounds that have antifungal activity has previously been demonstrated to occur earlier in SBSI, a wilt resistant cotton cultivar, than in the susceptible cultivar, Rowden. In this study we have used cloned probes to test the same cultivars for induced expression of genes from other known defense response pathways. The time course of mRNA appearance was followed for up to 4 days in stele tissues of control plants and in plants inoculated with conidia of the wilt pathogen, Verticillium dahliae . Among genes that code for enzymes in the phenylpropanoid defense pathway, phenylalanine ammonia-lyase mRNA was constitutively expressed. Levels of mRNA transcripts coding for chalcone synthase, an enzyme required for flavonoid biosynthesis, and for caffeic-O-methyltransferase, an enzyme used in synthesis of lignin, were greatly elevated in inoculated plants as compared to water treated controls. Greater and/or earlier initial response was detected in the wilt resistant cultivar than in the susceptible cultivar. Among genes coding for enzymes that can degrade fungal cell walls, equivalent levels of β-1,3 glucanase mRNA were present in both fungal and water-treated plants. Chitinase mRNA synthesis was strongly induced in inoculated plants. Levels of message continued to increase in Rowden, but diminished rapidly after 48–60 h in SBSI.

The Dc8 gene of carrot (Daucus carota L.) shows differential expression during embryo development. Changes in methylation patterns of a segment of about 500 bp (from base + 120 to base -446) of Dc8 allele 6 were investigated by treating genomic DNA, extracted from embryogenic callus at different stages of development, with sodium bisulfite to modify nonmethylated cytosines. Following asymmetric (strand-specific) amplification, base sequences for samples from each developmental stage were determined for each strand directly from the PCR products or from cloned PCR products. Different methylation patterns were detected in the two strands. The 5' to 3' sense (coding) strand was almost completely nonmethylated, whereas almost all the cytosines in the 3' to 5' (template) strand were methylated. By 71 days after transfer to embryo-inducing medium, few methylcytosines remained; those that were present were generally near the TATA box or in a region beyond -300. The cytosines that were methylated were not limited to CG or CNG sequences. The difference in the extent of methylation between the two complementary strands implies either that there is a mechanism for strand-specific methylation, or that complementary sequences can differ greatly in sensitivity to bisulfite treatment or PCR amplification.

Oligonucleotide primers made complementary to conserved sequences in phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes previously cloned from other species were used to amplify segments of the corresponding genes from sorghum. Greater than 70% base sequence identity with homologous genes confirmed that a 535 bp clone (PAL1-1) and a 620 bp clone (CHS2G) were derived from the coding regions of PAL and CHS respectively. When used to probe genomic digests, the two clones detected 3 and 5EcoRI fragments, respectively. One fragment for each probe was polymorphic in the parents of a mapping population, permitting a locus for each gene to be located on a sorghum RFLP linkage map. RNA gel blot analyses were performed on extracts from control seedlings and from seedlings challenged with either of two fungal sorghum pathogens or a pathogen of maize that elicits a hypersensitive response in sorghum. Although low levels of PAL and CHS mRNA transcripts were present constitutively in the controls, both rapidly accumulated to high levels following inoculation with the maize pathogen,Bipolaris maydis, and decreasing amounts of mRNA were apparent by 120 h post-inoculation. Since challenge withSporisorium reilianumwas by needle inoculation, an additional control of mock inoculations was included for this pathogen. However, no differences in the amount of mRNA for either gene were detected over the sampling period, whether samples from uninoculated, mock inoculated or spore-inoculated seedlings were tested. Differences in the timing and level of mRNA accumulation were detected in seedlings after inoculation withPeronosclerospora sorghi. While seedlings of both resistant and susceptible cultivars accumulated higher levels of PAL and CHS mRNA than uninoculated controls, the accumulation of mRNA in resistant cultivars was higher and longer lasting than that in susceptible cultivars.

DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.

The random amplified polymorphic DNA technique was used to find markers for a downy mildew resistance gene in sorghum. Of the 674 random primers screened for polymorphism, 2 amplified fragments were linked to a downy mildew resistance gene in sorghum line SC414. Utilization of an existing restriction fragment length polymorphism mapping population (IS3620C × BTx623) also revealed two markers that are linked to a different resistance gene in another sorghum line, BTx623.Key words: sorghum, downy mildew, RFLP, RAPD.

Sixty-two single-copy sorghum DNA clones were used to compare restriction fragment patterns of 53 sorghum accessions from Africa, Asia and the United States. Included were accessions from five morphological races of the cultivated subspecies bicolor, and four races of the wild subspecies verticilliflorum. From two to twelve alleles were detected with each probe. There was greater nuclear diversity in the wild subspecies (255 alleles in ten accessions) than in the domestic accessions (236 alleles in 37 accessions). Overall, 204 of the 340 alleles (60%) that were detected occurred in both subspecies. Phylogenetic analysis using parsimony separated the subspecies into separate clusters, with one group of intermediate accessions. Though exceptions were common, especially for the race bicolor, accessions classified as the same morphological race tended to group together on the basis of RFLP similarities. Selection for traits such as forage quality may have led to accessions genetically more similar to other races being classified as bicolors, which have a loose, small-grained panicle similar to wild races. Population statistics, calculated using four nuclear and four cytoplasmic probes that detect two alleles each, revealed a low but significant amount of heterozygosity, and showed little differentiation in alleles in the wild and cultivated subspecies. Outcrossing with foreign pollen appears to have been more important than migration via seed dispersal as a mechanism for gene flow between the wild and domestic accessions included in this study.

With rare exceptions, sorghum breeders have been highly successful in producing disease-resistant sorghums. Classical breeding techniques, including identification of resistant cultivars from large and readily available germplasm collections, crosses to elite but susceptible varieties, followed by backcrosses, selfing, and selection, have been used to develop disease-resistant breeding and commercial lines. However, for almost every disease, the breeding efforts must be continuous as new forms of the pathogens rapidly evolve to overcome resistance. Molecular tools that allow rapid and accurate tagging and identification of resistance genes permit, at the very least, the ability to use marker-assisted selection to combine different genes and to test the theory that stacking different genes for resistance will provide stable resistance. Knowledge of the nature and molecular functions of resistance genes promises much more: the ability to manipulate, alter, and enhance the signal transduction pathways that actually trigger host plant resistance.

The deliberate decision in the early 1940s to use Neurospora crassa in the Nobel Prize winning work that demonstrated an association between genes and enzymes initiated an avalanche of research now known as Molecular Genetics. Among the reasons Beadle and Tatum chose a fungus for that seminal work included the facts that while Neurospora is a eukaryote, it can be rapidly propagated from asexual conidia or mycelial fragments, with each culture providing millions of genetically identical haploid nuclei. Further, strains of opposite mating type can readily be crossed in order to analyze the inheritance pattern of mutant traits. When added to the fact that many fungi are pathogenic to plants and animals and contain much less DNA per genome than higher eukaryotes, it is somewhat surprising that the only fungal genome sequenced before the human genome was released in 2001 was that from a strain of Saccharomyces cerevisiae However, as rapid developments in technology have greatly reduced the expense of sequencing, fungal genomics has literally exploded. As of this writing, 353 fungal and 20 oomycete species are listed in the ‘genome completed’ section of The National Center for Biologic Information (NCBI). A ‘1,000’ Fungal Genomes Project initiated at the end of 2010.

A RFLP linkage map of sorghum composed principally of markers detected with sorghum low-copy-number nuclear DNA clones has been constructed. The map spans 1789 cMs and consists of 190 loci grouped into 14 linkage groups. The 10 largest linkage groups consist of from 10 to 24 markers and from 103 to 237 cMs, and the other 4 linkage groups consist of from 2 to 5 markers and from 7 to 62 cMs. The map was derived in Sorghum bicolor ssp. bicolor by analysis of a F2 population composed of 50 plants derived from a cross of IS 3620C, a guinea line, and BTx 623, an agronomically important inbred line derived from a cross between a zera zera (a caudatum-like sorghum) and an established kafir line. The restriction fragment length polymorphism (RFLP) frequency detected in this population using polymerase chain reaction (PCR)-amplifiable low-copy-number sorghum clones and five restriction enzymes was 51%. A minimal estimate of the number of clones that detect duplicate sequences is 11 %. Null alleles occurred at 13% of the mapped RFLP loci.

A recent outbreak of sorghum downy mildew (SDM) in Texas has led to the discovery of both metalaxyl fungicide resistance and a new pathotype, P6, in the causal organism Peronosclerospora sorghi. New and alternate sources of host plant resistance are needed for successful management of SDM. To identify sources of resistance, a total of 333 (242 minicore lines representing diverse germplasm from India, 67 commercial hybrids from Kansas, and 24 elite breeding lines from Texas) were inoculated in the greenhouse. Using an established sandwich inoculation technique, artificial inoculation of test lines with P. sorghi conidia, resulting in < 10% infection, were scored as “resistant.” Fifty-two minicore and 20 accessions from Kansas exhibited ≤ 10% infection and were selected as resistant. Out of 52 resistant minicore accessions, 28 were photoinsensitive. Eleven of 20 commercial hybrids from Kansas showed zero percent infection. Thirteen of 24 elite breeding lines from Texas were also resistant. In this study, resistance sources for the new P6 SDM pathotype were identified. The diversity among these materials is expected to provide different single-gene sources as well as quantitative sources of SDM resistance for use in breeding programs. Accepted for publication 13 January 2011. Published 23 March 2011.