Thomas Isakeit

Relationships Among Resistances to Fusarium and Aspergillus Ear Rots and Contamination by Fumonisin and Aflatoxin in Maize

ABSTRACT Fusarium verticillioides, F. proliferatum, and Aspergillus flavus cause ear rots of maize and contaminate the grain with mycotoxins (fumonisin or aflatoxin). The objective of this study was to investigate the relationships between resistance to Fusarium and Aspergillus ear rots and fumonisin and aflatoxin contamination. Based on a previous study of 143 recombinant inbred lines from the cross NC300 x B104, 24 lines with the highest and 24 lines with the lowest mean fumonisin concentration were selected for further evaluation. Paired plots of each line were inoculated with F. verticillioides and F. proliferatum or with A. flavus in replicated trials in 2004 and 2005 in Clayton, NC, and College Station, TX. The low-fumonisin group had significantly lower levels of fumonisin, aflatoxin, and Fusarium and Aspergillus ear rots. Across year-location environments, all four traits were significantly correlated; the genotypic correlation (r(G)) ranged from r(G) = 0.88 (aflatoxin and Aspergillus ear rot) to r(G) = 0.99 (Fusarium and Aspergillus ear rots). Quantitative trait loci (QTLs) were identified and their effects estimated. Two QTLs affected both toxin concentrations, one QTL affected both ear rots, and one QTL affected Aspergillus and Fusarium rots and fumonisin. These results suggest that at least some of the genes involved in resistance to ear rots and mycotoxin contamination are identical or genetically linked.

Natural infection of citronmelon with Acidovorax avenae subsp. citrulli.

Citronmelon fruits (Citrullus lanatus var. citroides (Bailey) Mansf.) with lesions were collected from a cowpea field in Frio County, TX, in July 1997. The lesions were circular, necrotic, or water-soaked, approximately 3 mm in diameter, and did not extend into the flesh of the fruit. Nonfluorescent, gram-negative bacteria were consistently isolated from lesions. Six representative strains were identified as Acidovorax avenae subsp. citrulli (Aac), using Biolog GN MicroPlates and the MicroLog data base release 3.50 (0.533 to 0.813 similarity). Aac causes leaf and fruit lesions (bacterial fruit blotch, BFB) on watermelon (C. lanatus (Thunb.) Matsum. & Nakai). Strains were tested for pathogenicity on watermelon seedlings (cv. Royal Sweet) by daubing bacterial suspensions (approximately 108 CFU/ml) onto cotyledons of 1-week-old seedlings. Water soaking of cotyledons, followed by necrosis and seedling death, occurred within 5 days. These symptoms were indistinguishable from those caused by watermelon strains of Aac. Bacteria were reisolated from symptomatic seedlings. The source of the infection is not known. Watermelons had been grown in this field in 1996, but no BFB symptoms were observed. Citron fruit infected with Aac were found in nearby watermelon fields where BFB was present; the closest field was 50 m from the cowpea field. These observations suggest that citronmelon, a common weed in south Texas, has the potential to perpetuate Aac. This is the first documentation of a naturally occurring infection of citronmelon with Aac.

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton

Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC.

Genome Wide Association Study for Drought, Aflatoxin Resistance, and Important Agronomic Traits of Maize Hybrids in the Sub-Tropics

The primary maize (Zea mays L.) production areas are in temperate regions throughout the world and this is where most maize breeding is focused. Important but lower yielding maize growing regions such as the sub-tropics experience unique challenges, the greatest of which are drought stress and aflatoxin contamination. Here we used a diversity panel consisting of 346 maize inbred lines originating in temperate, sub-tropical and tropical areas testcrossed to stiff-stalk line Tx714 to investigate these traits. Testcross hybrids were evaluated under irrigated and non-irrigated trials for yield, plant height, ear height, days to anthesis, days to silking and other agronomic traits. Irrigated trials were also inoculated with Aspergillus flavus and evaluated for aflatoxin content. Diverse maize testcrosses out-yielded commercial checks in most trials, which indicated the potential for genetic diversity to improve sub-tropical breeding programs. To identify genomic regions associated with yield, aflatoxin resistance and other important agronomic traits, a genome wide association analysis was performed. Using 60,000 SNPs, this study found 10 quantitative trait variants for grain yield, plant and ear height, and flowering time after stringent multiple test corrections, and after fitting different models. Three of these variants explained 5–10% of the variation in grain yield under both water conditions. Multiple identified SNPs co-localized with previously reported QTL, which narrows the possible location of causal polymorphisms. Novel significant SNPs were also identified. This study demonstrated the potential to use genome wide association studies to identify major variants of quantitative and complex traits such as yield under drought that are still segregating between elite inbred lines.

Characterization of genetic diversity and linkage disequilibrium of ZmLOX4 and ZmLOX5 loci in maize.

Maize (Zea mays L.) lipoxygenases (ZmLOXs) are well recognized as important players in plant defense against pathogens, especially in cross kingdom lipid communication with pathogenic fungi. This study is among the first to investigate genetic diversity at important gene paralogs ZmLOX4 and ZmLOX5. Sequencing of these genes in 400 diverse maize lines showed little genetic diversity and low linkage disequilibrium in the two genes. Importantly, we identified one inbred line in which ZmLOX5 has a disrupted open reading frame, a line missing ZmLOX5, and five lines with a duplication of ZmLOX5. Tajimas D test suggests that both ZmLOX4 and ZmLOX5 have been under neutral selection. Further investigation of haplotype data revealed that within the ZmLOX family members only ZmLOX12, a monocot specific ZmLOX, showed strong linkage disequilibrium that extends further than expected in maize. Linkage disequilibrium patterns at these loci of interest are crucial for future candidate gene association mapping studies. ZmLOX4 and ZmLOX5 mutations and copy number variants are under further investigation for crop improvement.

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

Tomato yellow leaf curl virus in Tomato in Texas, Originating from Transplant Facilities

Tomato yellow leaf curl virus (TYLCV), a monopartite virus in the genus Begomovirus (family, Geminiviridae) from the Middle East, is one of the most damaging whitefly-transmitted viruses of tomato (Lycopersicon esculentum) worldwide. TYLCV was first identified in the United States in 1997 in Florida (4), and most recently, in the Pacific Coast states of Mexico where fresh market tomatoes are grown for the U.S. market (1). During September 2006, tomatoes grown from transplants in Waller County, TX exhibited shortened internodes, stunting and puckering of leaflets, green vein banding, and diffuse chlorosis. The disease incidence in two fields (4 ha total) was 95% and yield was substantially reduced. Many of the transplants were symptomatic at planting. The transplants originated from two facilities in Hidalgo County, TX. Both facilities had experienced heavy infestations of the whitefly, Bemisia tabaci (Genn.), during transplant production. At the same time, transplants produced in Uvalde and Bexar counties, TX, where whitefly infestations were also prevalent, had similar virus symptoms. Total DNA was extracted from the leaves of symptomatic tomato plants from 10 samples from these four counties and amplified by PCR (2). DNA samples from Waller, Hidalgo, and Uvalde counties were cloned, and a partial fragment of the viral coat protein gene (core Cp) was sequenced. BLAST analysis of the core Cp sequences of each sample confirmed the presence of TYLCV. No other begomovirus was detected, and all attempts to amplify a bipartite begomovirus by PCR using degenerate DNA-B specific primers (3) were unsuccessful. The full-length TYLCV DNA was amplified from three samples using the rolling circle amplification method as described (1), cloned, and the sequences were determined. The three sequences shared 99.6 to 100% nt identity and so only one sequence was deposited in the NCBI GenBank database (Accession No. EF110890) (1). Analysis of the complete genome nucleotide sequence corroborated TYLCV identity predicted by core Cp analysis that was 98.1% identical with TYLCV from Egypt (GenBank Accession No. AY594174) and Spain (GenBank Accession No. AJ489258), 97.6% with TYLCV from Mexico (GenBank Accession No. DQ631892), and 96.5% with TYLCV-Is (GenBank Accession No. X15656). Additionally, a Southern blot with TYLCV as the probe detected replicating (double-stranded) TYLCV DNA in all samples consisting of three plants from Uvalde County and 21 plants from Bexar County. To our knowledge, this is the first report of TYLCV in Texas that occurred in two transplant production areas approximately 400 km apart. Transplants produced in Uvalde and Bexar counties were planted there, while Hidalgo County transplants were shipped outside of the usual range of the whitefly. Hidalgo County has a subtropical climate, which can allow overwintering of TYLCV and the whitefly vector, allowing the establishment and spread of this virus in the future. References: (1) J. K. Brown and A. M. Idris. Plant Dis. 90:1360, 2006. (2) J. K. Brown et al. Arch. Virol. 146:1581, 2001. (3) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998. (4) J. E. Polston et al. Plant Dis. 83:984, 1999.

Effect of temperature and relative humidity on sorghum ergot development in northern Mexico

Trials were planted at Rio Bravo, Tamaulipas, Mexico, during 2002 and 2003 with the objective of determining the relationship between sorghum ergot severity and weather factors, and to develop a risk assessment model. Six sorghum hybrids and three male-sterile genotypes were planted every month from January to October. At anthesis initiation, inoculation was conducted using a local isolate of Claviceps africana. Among hybrids, there was a negative relationship between ergot severity and maximum and minimum temperatures, with the highest significant correlation of —0.71 from 7 to 9 days before anthesis. During this period, minimum temperatures above 10°C increased the risk of ergot development, whereas minimum temperatures above 22.5°C prevented ergot development. In male-sterile plants, ergot was negatively related to maximum temperature after anthesis, with ergot being observed at maximum temperatures up to 38°C. Minimum relative humidity showed a positive and significant correlation with ergot severity. Values of minimum relative humidity above 30% during anthesis promoted infection. Surface response regression models were developed for the effects of minimum relative humidity and minimum and maximum temperature during the infection process on ergot severity.