Renée S. Arias

Somatic mutation‐mediated evolution of herbicide resistance in the nonindigenous invasive plant hydrilla (Hydrilla verticillata)

Hydrilla (Hydrilla verticillata L.f. Royle) was introduced to the surface water of Florida in the 1950s and is today one of the most serious aquatic weed problems in the USA. As a result of concerns associated with the applications of pesticides to aquatic systems, fluridone is the only USEPA‐approved chemical that provides systemic control of hydrilla. After a decrease in fluridones efficacy at controlling hydrilla, 200 Florida water bodies were sampled to determine the extent of the problem and the biological basis for the reduced efficacy. Our studies revealed that hydrilla phenotypes with two‐ to six‐fold higher fluridone resistance were present in 20 water bodies. Since fluridone is an inhibitor of the enzyme phytoene desaturase (PDS), the gene for PDS (pds) was cloned from herbicide‐susceptible and ‐resistant hydrilla plants. We report for the first time in higher plants three independent herbicide‐resistant hydrilla biotypes arising from the selection of somatic mutations at the arginine 304 codon of pds. The three PDS variants had specific activities similar to the wild‐type enzyme but were two to five times less sensitive to fluridone. In vitro activity levels of the enzymes correlated with in vivo resistance of the corresponding biotypes. As hydrilla spread rapidly to lakes across the southern United States in the past, the expansion of resistant biotypes is likely to pose significant environmental challenges in the future.

Susceptibility of isofamilies of Spodoptera frugiperda (Lepidoptera: Noctuidae) to Cry1Ac and Cry1Fa proteins of Bacillus thuringiensis.

Abstract. The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the most important insect pests on the American continent. Its control has relied primarily on multiple applications of insecticides that can amount to 1,000 g of active ingredient per hectare on some of approximately 30 crops the insect damages. The use of genetically engineered crops that express Bacillus thuringiensis (Bt) Berliner toxins, Bt-corn, Zee meys L.; and Bt-cotton, Gossypium hirsutum L.; are other ways to control this insect. However, fall armyworm is one of the Lepidoptera species least susceptible to Bt proteins, and a case of high tolerance to Bt-corn has already being reported. We found the susceptibility to Cry1Ac and Cry1Fa proteins of Bt in 133 isofamilies from five regions of three countries was similar to the susceptibility of two Bt-susceptible laboratory colonies to these proteins. Four isofamilies from Puerto Rico were very tolerant to Cry1Fa and not so tolerant to Cry1Ac. Two of the four isofamilies were backcrossed with a Bt-susceptible laboratory colony and their progeny was as susceptible to both Bt proteins as was the Bt-susceptible colony, indicating that resistance to Bt is a recessive trait.

Optimized construction of microsatellite-enriched libraries

The construction of microsatellite‐enriched libraries is an indispensable tool to search for molecular markers as complete genome sequences are still not available for the majority of species of interest. Numerous protocols are available in the literature for the construction of these libraries; however, sometimes their low efficiency or lack of optimization in the protocols can restrict their efficacy. We have designed and tested various adapters and ligation methods; we also tested oligo‐repeat combinations and hybridization temperatures, and created libraries with this new protocol for four organisms: Ipomoea batatas (L.) Lam, Chionanthus retusus Lindley & Paxton, Rotylenchulus reniformis Linford & Olivera and Puccinia kuehnii W. Krüger. The number of microsatellites detected for these species ranged from 2494 to 3919 per Mb of nonredundant sequence, that was 0.86 and 1.53 microsatellites per contig, with 37–66% of di‐nucleotide motifs and 21–49% of tri‐ to octa‐nucleotide repeats combined. A simplified protocol is provided for the successful generation of SSR‐enriched libraries.

First Microsatellites from Spodoptera frugiperda (Lepidoptera: Noctuidae) and Their Potential use for Population Genetics

ABSTRACT This is the first report of sequence-specific microsatellite markers (simple sequence repeats [SSRs]) of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), an economically important pest of crops on the Americas. We isolated 192 microsatellite markers by using pyrosequencing and screened 15 individuals from eight isofamilies collected from three geographical areas: Puerto Rico (PR), Texas (TX), and Mississippi (MS). Isofamilies resistant to Cry toxins from Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae) also were included. Cluster analysis was performed to determine the potential use of these SSRs in discriminating populations, and colonies were grouped with a reliability of 100% estimated by bootstrap. In this analysis, colonies from TX grouped away from those from PR, but the two MS isofamilies grouped with TX and PR separately. Genetic distance within isofamilies ranged between 0.22 and 0.56, and the minimum distance between isofamilies was 0.83. Unique pattern informative combination (UPIC) scores were calculated, and the 80 SSR markers that had UPIC scores of ≥1 are listed according to their discriminating potential. UPIC scores allow reducing costs by choosing fewer and highly informative markers for future studies. From the best 125 markers, 103 had a maximum of two alleles per sample, making them ideal candidates for population genetic studies. BLAST screening of the sequences points to potential biological meaning of marker polymorphisms. The percentage of alleles shared by the three geographic areas was 14%. The markers reported will significantly enrich the pool of molecular markers available for S. frugiperda. In addition, they could be used for monitoring migration of populations, in the development of biocontrol agents and for management practices in general.

Genome Sequences of Eight Aspergillus flavus spp. and One A. parasiticus sp., Isolated from Peanut Seeds in Georgia

ABSTRACT Aspergillus flavus and A. parasiticus fungi produce carcinogenic mycotoxins in peanut seeds, causing considerable impact on both human health and the economy. Here, we report nine genome sequences of Aspergillus spp., isolated from Georgia peanut seeds in 2014. The information obtained will lead to further biodiversity studies that are essential for developing control strategies.

UPIC: Perl scripts to determine the number of SSR markers to run.

We introduce here the concept of Unique Pattern Informative Combinations (UPIC), a decision tool for the cost-effective design of DNA fingerprinting/genotyping experiments using simple-sequence/tandem repeat (SSR/STR) markers. After the first screening of SSR-markers tested on a subset of DNA samples, the user can apply UPIC to find marker combinations that maximize the genetic information obtained by a minimum or desirable number of markers. This allows a cost-effective planning of future experiments. We have developed Perl scripts to calculate all possible subset combinations of SSR markers, and determine based on unique patterns or alleles, which combinations can discriminate among all DNA samples included in a test. This makes UPIC an essential tool for optimizing resources when working with microsatellites. An example using real data from eight markers and 12 genotypes shows that UPIC detected groups of as few as three markers sufficient to discriminate all 12- DNA samples. Should markers for future experiments be chosen based only on polymorphism-information content (PIC), the necessary number of markers for discrimination of all samples cannot be determined. We also show that choosing markers using UPIC, an informative combination of four markers can provide similar information as using a combination of six markers (23 vs. 25 patterns, respectively), granting a more efficient planning of experiments. Perl scripts with documentation are also included to calculate the percentage of heterozygous loci on the DNA samples tested and to calculate three PIC values depending on the type of fertilization and allele frequency of the organism. Availability Perl scripts are freely available for download from http://www.ars.usda.gov/msa/jwdsrc/gbru.

Genomics of Tropical Fruit Tree Crops

The genetic improvement of tropical fruit trees is limited when compared to progress achieved in temperate fruit trees and annual crops. Tropical fruit tree breeding programs require significant resources to develop new cultivars that are adapted to modern shipping and storage requirements. The use of molecular markers in tropical fruit tree breeding is greatly assisting in solving a number of difficult challenges for breeders such as the development of complex family structures for recombination mapping and for recurrent selection. A review of the literature on molecular markers development and new techniques for increasing single-nucleotide polymorphic markers is discussed. The development of marker-assisted breeding for these tropical tree crops is also discussed.

Draft Genome Sequence of Cercospora arachidicola,Causal Agent of Early Leaf Spot in Peanuts

ABSTRACT Cercospora arachidicola, causal agent of early leaf spot, is an economically important peanut pathogen. Lack of genetic information about this fungus prevents understanding the role that potentially diverse genotypes may have in peanut breeding programs. Here, we report for the first time a draft genome sequence of C. arachidicola.

Suppression of aflatoxin production in Aspergillus species by selected peanut (Arachis hypogaea) stilbenoids

Aspergillus flavus is a soil fungus that commonly invades peanut seeds and often produces carcinogenic aflatoxins. Under favorable conditions, the fungus-challenged peanut plant produces and accumulates resveratrol and its prenylated derivatives in response to such an invasion. These prenylated stilbenoids are considered peanut antifungal phytoalexins. However, the mechanism of peanut-fungus interaction has not been sufficiently studied. We used pure peanut stilbenoids arachidin-1, arachidin-3, and chiricanine A to study their effects on the viability of and metabolite production by several important toxigenic Aspergillus species. Significant reduction or virtually complete suppression of aflatoxin production was revealed in feeding experiments in A. flavus, Aspergillus parasiticus, and Aspergillus nomius. Changes in morphology, spore germination, and growth rate were observed in A. flavus exposed to the selected peanut stilbenoids. Elucidation of the mechanism of aflatoxin suppression by peanut stilbenoids could provide strategies for preventing plant invasion by the fungi that produce aflatoxins.

Status of Macrophomina phaseolina on Strawberry in California and Preliminary Characterization of the Pathogen

ABSTRACT Macrophomina crown and root rot has become a significant soil-borne disease issue in California. For many locations in the state, the disease is associated with fields that are no longer pre-plant, flat field fumigated with methyl bromide + chloropicrin. Inoculation experiments indicated that some differences in strawberry cultivar susceptibility to Macrophomina phaseolina were seen a short time after the inoculation, but as disease progressed such differences did not persist. Preliminary characterization studies of Macrophomina phaseolina isolates from strawberry indicated that such isolates may have a host preference for strawberry. Macrophomina phaseolina isolates from watermelon, thyme, and apple failed to cause disease in strawberry. Five cover crop species, which can be rotated with strawberry, did not develop disease when inoculated with strawberry isolates. In preliminary analysis using simple sequence repeat markers, isolates obtained from strawberry formed a separate group compared to isolates recovered from other known Macrophomina phaseolina hosts.