Louis K. Prom

Response of eight sorghum cultivars inoculated with Fusarium thapsinum, Curvularia lunata, and a mixture of the two fungi

Abstract Field studies were conducted in 2000 and 2001 to investigate effects of two common grain mold fungi, Fusarium thapsinum, Curvularia lunata and a mixture of the two pathogens, on grain mold severity and seed germination in eight sorghum cultivars with different levels of vulnerability to grain mold. Significant effects due to cultivar, fungal treatment and their interaction were observed for grain mold severity (GMS) and for germination. Fungal treatments increased GMS and reduced seed germination in all cultivars. Sureno was the most resistant cultivar evaluated. All other cultivars exhibited susceptible reactions. The type of grain mold fungi present in the field influenced the level of GMS, since cultivars responded differently to inoculation with individual or multiple fungal species. Germination was significantly reduced by all fungal treatments. C. lunata had the most negative effect on germination in 2000 while F. thapsinum produced the most reduction in germination in 2001. These pathogens caused significant reductions in the germination rate, even though GMS ratings on inoculated panicles were low.

Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

BackgroundA recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, Peronosclerospora sorghi. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.ResultsAmong the 55 primers pairs designed from clones from pathotype 3 of P. sorghi, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from P. sorghi, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (P. maydis & P. philippinensis), sugar cane (P. sacchari), pearl millet (Sclerospora graminicola) and rose (Peronospora sparsa) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to P. philippinensis (one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 Peronosclerospora, Peronospora and Sclerospora spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of Peronosclerospora sorghi in cluster I, five isolates of P. maydis and three isolates of P. sacchari in cluster II and five isolates of Sclerospora graminicola in cluster III).ConclusionTo our knowledge, this is the first attempt to extensively develop SSR markers from Peronosclerospora genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.

Expression of pathogenesis-related protein PR-10 in sorghum floral tissues in response to inoculation with Fusarium thapsinum and Curvularia lunata

Differences in grain mould disease levels among different sorghum varieties grown in the same environment imply that host genes play a role in controlling disease severity. The fungi most often recovered from naturally infected sorghum grain, Fusarium thapsinum and Curvularia lunata, were used to inoculate a set of resistant and susceptible cultivars at anthesis in both field and glasshouse trials. In the field, 12 cultivars were inoculated with a mixture of F. thapsinum and C. lunata and, in the glasshouse, individual panicles from four selected cultivars were inoculated with spore suspensions of C. lunata, F. thapsinum, a mixture of the two or water to serve as a control. Based on grain mould severity ratings and germination tests on the seed from the field trial, Tx2911, Sureno, SC719-11E and SC650-11E displayed a high level of resistance to grain mould. To determine whether resistant and susceptible lines differed in response to the pathogens, PR-10 mRNA levels were measured using real-time reverse transcriptase-polymerase chain reaction. PR-10 is a protein with antifungal properties that has been associated with defence responses in sorghum and other plant species. In field tests, most, but not all, cultivars showed significant induction of normalized relative quantities of PR-10 after dual inoculation with spores of both C. lunata and F. thapsinum. Under glasshouse-controlled conditions, glumes of inoculated plants showed the clear induction of PR-10 mRNA, and the response was greater in resistant (Tx2911 and Sureno) than in susceptible (RTx430 and SC170-6-17) cultivars. Inoculation with spores from a single mould-inducing pathogen generally induced greater responses than when spores were combined. For RTx430, SC170-6-17 and Sureno, the response to C. lunata was greater, whereas Tx2911 showed a stronger response to F. thapsinum. The results indicate that the induction of PR-10 in sorghum glumes may be a factor useful in breeding programmes designed to combine multiple factors for resistance.

The Effects of Fusarium thapsinum, Curvularia lunata, and Their Combination on Sorghum Germination and Seed Mycoflora

Abstract Grain mold, caused by several species of fungi, reduces grain yield and quality. This study examined the effect of the two most common grain mold fungi, Fusarium thapsinum and Curvularia lunata, inoculated singly and together on germination and seed mycoflora. Germination rates for the different cultivars have been reported in details previously. C. lunata was the most frequently isolated fungal species followed by F. thapsinum and F. semitectum. Non-inoculated control samples had Alternaria spp. and F. semitectum as the most frequently recovered fungal species followed by C. lunata. There were highly significant negative correlations between germination and C. lunata (r = - 0.44 and P < 0.01) in 2000 and between germination and F. thapsinum (r = −0.52 and P < 0.01) in 2001. Significant negative correlations between F. semitectum andF. thapsinum, and highly significant negative correlations between F. thapsinum and C. lunata were observed. Data from this study indicate that the fungal species present, the environment, and the sorghum cultivar all influence seed mycoflora.

Viability of Claviceps africana Spores Ingested by Adult Corn Earworm Moths, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

Abstract A study was conducted in College Station, TX, to determine the viability of Claviceps africana spores in the digestive tract of adult corn earworm moths, Helicoverpa zea (Boddie). Both sexes were exposed to ergot-infected sorghum panicles for 30 min, and spores were recovered from excreta of the moths at 24-, 48-, and 72-h intervals after feeding. Recovered spores were quantified, and viability was determined by the germination rate of macroconidia. Nearly a 100-fold greater concentration of spores was recovered from female excreta at the three time intervals compared with male excreta. Concentration of spores in female and male excreta was greatest at 24 h, with a significant reduction at the later time intervals. Spore germination rates for both sexes were greater at 24 h, with survival being significantly reduced at the 72-h interval. Spores in female excreta survived longer than those from male excreta. Spore survival over time was significantly reduced in male excreta. Spore concentration and survival were greater from female excreta, which is key, because egg-laying activities on sorghum panicles intensify during flowering, and this source of ergot spores could contribute to the spread of the disease. This study demonstrates that corn earworm moths can internally carry viable ergot spores for several days and can act as primary dispersal agents for the fungus. This is important because contaminated moths migrating from areas in Mexico and southern Texas where ergot is endemic could transmit and spread the disease to other sorghum growing regions of the United States.

Transmission of Claviceps africana Spores from Diseased to Non-Infected Sorghum by Corn Earworm Moths, Helicoverpa zea

ABSTRACT The potential of corn earworm moths Helicoverpa zea, an important pest of sorghum in Texas, as a passive vector of sorghum ergot, Claviceps africana was assessed. Laboratory-reared (females and males) and field-moths trapped in sex pheromone traps were confined in screen cages with potted male-sterile sorghum line AT×623 that had been artificially infected with sorghum ergot and was producing massive amounts of honeydew that contained spores of the fungus. In the cages, moths were observed feeding on the honeydew. When laboratory-reared moths contaminated with C. africana spores were released for 12 or 48 h in cages containing healthy greenhouse- or field-grown male-sterile sorghum plants at anthesis, the levels of ergot severities on the panicles ranged from 0.5–90%, per panicle. The levels of ergot infection on greenhouse- and field-grown AT×623 exposed to ergot contaminated field-trapped males for 48 h ranged from 0–81.7%. Mean external contamination of laboratory-reared and field-trapped moths placed in a soapy wash was 1.4 × 105 and 4.7 ×104 conidia/ml, respectively. This study demonstrated that contaminated adult corn earworm could transmit the sorghum ergot from diseased to healthy panicles when environmental conditions are favorable for infection. Thus, migrating moths and other highly mobile pest species have the potential to transport C. africana over relatively large distances.

Virulence and Molecular Genotyping Studies of Sporisorium reilianum Isolates in Sorghum

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.

Survival of Claviceps africana Within Sorghum Panicles at Several Texas Locations

Survival of the sorghum ergot fungus, Claviceps africana, based on pathogenicity of recovered macroconidia used to inoculate sorghum (Sorghum bicolor), was measured in 2000 over the course of the year at five locations in Texas representing three climates. The experiment was repeated in 2001. Sphacelia associated with infected sorghum panicles were placed in nylon mesh bags and either buried at a 10-cm depth, placed on the soil surface, or suspended 61 cm above the ground. Samples were recovered after 4, 8, and 12 months and assessed for pathogenicity of surviving macroconidia by macerating tissue in water and spraying it onto panicles of flowering male-sterile sorghum in the greenhouse. Survival of ergot macroconidia in recovered panicles declined at all locations after the first 4 months that panicles were left in the field. The decline in viability during this period was greater in 2001 than in 2000. In 2000, survival after 4 months was greatest at Lubbock and Bushland, which have a continental steppe climate, than at the other three Texas locations, Weslaco and Corpus Christi, which have a subtropical subhumid climate, and College Station, which has a subtropical humid climate. However, this difference in survival was not as pronounced in 2001. Additionally, after 8 months, survival levels at all locations were similar. At the end of 12 months, infective macroconidia were found only at Lubbock in 2000, and only at Lubbock and College Station in 2001. Ergot macroconidia can survive in all major sorghum production areas of Texas; thus, conidia would not need to move long distances in order to initiate an epiphytotic.

Laboratory, greenhouse, and field assessment of fourteen fungicides for activity against Claviceps africana, causal agent of sorghum ergot

Commercial formulations of 14 fungicides representing seven chemical classes were assessed in vitro and in vivo for activity against Claviceps africana, causal agent of sorghum ergot. All fungicides markedly reduced spore germination in vitro on water agar, with effective concentration (EC)50 values (based on active ingredient) that ranged from 0.01 μg ml-1 for thiophanatemethyl and trifloxystrobin to 1.18 μg ml-1 for cyprodinil. In vivo assays included applications of fungicide on full bloom panicles of a male-sterile line, ATx623, and then inoculating panicles with a conidial suspension immediately after the fungicide had dried. In greenhouse trials and in the 2000 field evaluation, a single application of propiconazole, tebuconazole, triadimefon, myclobutanil, or azoxystrobin at a rate of 25 μg ml-1 markedly suppressed ergot severity on ATx623. In the 2001 field evaluation, tebuconazole applied at 25 μg ml-1 was most effective in reducing levels of ergot severity. Ergot control was greater when fungicides were applied at 25 μg ml-1 than at 5 μg ml-1. Generally, triazole and strobilurin classes of fungicide were more effective in controlling ergot than benzimidazole, anilinopyrimidine, ethylenebisdithiocar-bamate (EBDC), pyridinamine, and organometallic classes. At the two fungicide rates used in this study, ergot severity increased between 7 and 10 days after inoculation. Results showed that in vitro fungicide screening by measuring spore germination inhibition was not a good predictor of performance in the field. Results from the field trials suggest that control, particularly under ergot-favorable environments, may require higher rates than used in this study.

A Pictorial Technique for Mass Screening of Sorghum Germplasm for Anthracnose (Colletotrichum sublineolum) Resistance

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