Fitness Attributes of Pythium aphanidermatum with Dual Resistance to Mefenoxam and Fenamidone.

Pythium aphanidermatum is the predominant species causing Pythium root rot on commercially grown poinsettias in North Carolina. Resistance to mefenoxam is common in populations of P. aphanidermatum but resistance to fenamidone and other quinone outside inhibitor fungicides has only just been reported in greenhouse floriculture crops. The in vitro sensitivity to the label rate of mefenoxam (17.6 µl active ingredient [a.i.]/ml) and fenamidone (488 µl a.i./ml) was determined for 96 isolates of P. aphanidermatum. Isolates were assigned to four fungicide phenotypes: mefenoxam-sensitive/fenamidone-sensitive (MefS, FenS), mefenoxam-sensitive/fenamidone-insensitive (MefS, FenR), mefenoxam-insensitive/fenamidone-sensitive (MefR, FenS), and mefenoxam-insensitive/fenamidone-insensitive (MefR, FenR). In all, 58% of isolates were insensitive to one (MefR, FenS = 36% and MefS, FenR = 16%) or both fungicides (MefR, FenR = 6%). A single point mutation in the cytochrome b gene (G143A) was identified in fenamidone-insensitive isolates. Mycelial growth rate at three temperatures (20, 25, and 30°C), in vitro oospore production, and aggressiveness on poinsettia were evaluated to assess relative fitness of sensitive and insensitive isolates. Isolates with dual insensitivity to mefenoxam and fenamidone had reduced radial hyphal growth at 30°C and produced fewer oospores than isolates sensitive to one or both fungicides. Isolates sensitive to both fungicides produced greater numbers of oospores. Aggressiveness on poinsettia varied by isolate but fungicide phenotype was not a good predictor of aggressiveness. These results suggest that populations of P. aphanidermatum with dual resistance to mefenoxam and fenamidone may be less fit than sensitive populations under our imposed experimental conditions but populations of P. aphanidermatum should continue to be monitored in poinsettia production systems for mefenoxam and fenamidone insensitivity.

Population genetic analysis of Tomato spotted wilt virus on peanut in North Carolina and Virginia.

Exploring the genetic diversity and evolutionary history of plant viruses is critical to understanding their ecology and epidemiology. In this study, maximum-likelihood and population genetics-based methods were used to investigate the population structure, genetic diversity, and sources of genetic variation in field isolates of Tomato spotted wilt virus (TSWV) from peanut in North Carolina and Virginia. Selected regions of the nucleocapsid, movement, and RNA-dependent RNA polymerase genes were amplified and sequenced to identify haplotypes and infer genetic relationships between isolates of TSWV with heuristic methods. The haplotype structure of each locus consisted of 1 or 2 predominant haplotypes and >100 haplotypes represented by a single isolate. No specific haplotypes were associated with geographic area, peanut cultivar, or year of isolation. The population was panmictic at the regional level and high levels of genetic diversity were observed among isolates. There was evidence for positive selection on single amino acids in each gene on a background of predominant purifying selection acting upon each locus. The results of compatibility analyses and the persistence of specific gene sequences in isolates collected over three field seasons suggest that recombination was occurring in the population. Estimates of the population mutation rate suggest that mutation has had a significant effect on the shaping of this population and, together with purifying selection, these forces have been the predominant evolutionary forces influencing the TSWV population in peanut in North Carolina and Virginia.

First Report of Root Rot Caused by Binucleate Rhizoctonia Anastomosis Group F on Musa spp.

A research program was initiated at the University of Georgia in 2003 to identify banana cultivars suitable for production in the coastal and southern areas of the state. During a root disease survey conducted in October 2007 on bananas (Musa spp.) grown at the University of Georgia Bamboo Farm and Coastal Gardens in Savannah, GA, root lesions and root rot were observed on banana cvs. Gold Finger, Kandarian, and Manzano. Root lesions were dark brown to black and irregular in shape, with partial or entire roots affected. Lateral roots and outer layers of cord roots (roots arising from interior layers of the corm) of infected plants were blackened and rotted. Diseased root samples were collected from three plants of each cultivar, surface sterilized with 0.6% sodium hypochlorite, and placed on tannic acid benomyl agar (TABA). Pure cultures of the fungus consistently associated with diseased tissue were obtained by subculturing hyphal tips on TABA. Mycelia of the fungus on potato dextrose agar (PDA) were light to deep brown and the hyphae tended to branch at right angles. A septum was present in each hyphal branch near the point of origin and a slight constriction at the branch was observed. The hyphae of two isolates were stained with 0.6% phenosafranin and 3% KOH and binucleate hyphal cells were observed. On the basis of these morphological features, the isolates appeared to be binucleate Rhizoctonia anamorphs (teleomorph Ceratobasidium Rogers). For molecular identification, the internal transcribed spacer (ITS) regions and the 5.8S gene from rDNA of the isolates were cloned and sequenced (GenBank Accession No. HQ168370). The ITS regions (775 bp) were 100% identical between the two isolates and 99% identical to Ceratobasidium sp. AG-F strain SIR-1 isolated from sweet potato in Japan (GenBank Accession No. AF354085). The anastomosis group of the isolates was confirmed by pairing with strain SIR-1 on PDA. On the basis of morphological and molecular characteristics and the anastomosis assay, the two isolates were identified as a Ceratobasidium sp. AG-F (1-3). Pathogenicity assays were conducted by inoculating banana plants (cv. Golden pillow, synonym = Manzano) grown in pots under greenhouse conditions (25 to 27°C). Twenty wheat seeds infested with each isolate were placed uniformly around each plant at a depth of 10 cm in the soil. The plants were incubated in the greenhouse and the roots were examined 2 months after inoculation. Brown-to-black lesions and root rot, identical to symptoms associated with field banana roots, were observed on all inoculated plants but not on the noninoculated control plants. The fungus was reisolated from affected root samples and the identity was confirmed by morphological and molecular characteristics and the anastomosis assay. To our knowledge, this is the first report of banana root rot caused by binucleate Rhizoctonia anastomosis group F. With the increased interest in producing bananas for food and ornamental purposes, the occurrence of Ceratobasidium root rot on bananas needs to be considered when designing disease management programs and searching for suitable cultivars for banana production. References: (1) L. L. Burpee et al. Mycologia 70:1281, 1978. (2) D. González et al. Mycologia 93:1138, 2001. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN. 1991.

An Assessment of the Genetic Diversity in a Field Population of Phytophthora nicotianae with a Changing Race Structure.

One hundred fifty-three isolates of Phytophthora nicotianae that were collected over a 4-year period from a single field were subjected to amplified fragment length polymorphism (AFLP) analysis to investigate the effect of different types of resistance in tobacco (Nicotiana tabacum) on genetic diversity in the pathogen population. No race 1 isolates were detected in the field prior to initiating the study, but the race was present in multiple plots by the end of the 4-year period. There were 102 race 0 isolates and 51 race 1 isolates characterized. Seventy-six of the 153 isolates had a unique AFLP profile, whereas the remaining 77 isolates were represented by 27 AFLP profiles shared by at least two isolates. Isolates of both races were found in both the unique and shared AFLP profile groups. Twenty-three of the AFLP profiles were detected in multiple years, indicating a clonal component to the pathogen population. Race 1 isolates that were detected over multiple years were always obtained from the same plot. No race 1 profile was found in more than one plot, confirming the hypothesis that the multiple occurrences of the race throughout the field were the result of independent events and not pathogen spread. Three identical race 0 AFLP profiles occurred in noncontiguous plots, and in each case, the plots contained the same partially resistant variety. Cluster analysis provided a high level of bootstrap support for 41 isolates in 19 clusters that grouped primarily by race and rotation treatment. Estimates of genetic diversity ranged from 0.365 to 0.831 and varied depending on tobacco cultivar planted and race. When averaged over all treatments, diversity in race 1 isolates was lower than in race 0 isolates at the end of each season. Deployment of single-gene resistance initially decreased genetic diversity of the population, but the diversity increased each year, indicating the pathogen was adapting to the host genotypes deployed in the field.

Highly polymorphic in silico-derived microsatellite loci in the potato-infecting fungal pathogen Rhizoctonia solani anastomosis group 3 from the Colombian Andes.

Fourteen polymorphic microsatellite DNA markers derived from the draft genome sequence of Rhizoctonia solani anastomosis group 3 (AG-3), strain Rhs 1AP, were designed and characterized from the potato-infecting soil fungus R. solani AG-3. All loci were polymorphic in two field populations collected from Solanum tuberosum and S. phureja in the Colombian Andes. The total number of alleles per locus ranged from two to seven, while gene diversity (expected heterozygosity) varied from 0.11 to 0.81. Considering the variable levels of genetic diversity observed, these markers should be useful for population genetic analyses of this important dikaryotic fungal pathogen on a global scale.

Genetic Characterization of Binucleate Rhizoctonia Species Causing Web Blight on Azalea in Mississippi and Alabama.

Web blight on containerized azalea is an annual problem for commercial nurseries during summer months in the southern United States. Losses to web blight are associated with the cost of fungicide applications, delayed marketing of diseased plants, and plant death. Two hundred and eleven isolates of binucleate Rhizoctonia were recovered from azalea leaves with web blight symptoms from two nurseries in Mississippi and Alabama over 3 years. The internal transcribed spacer region (ITS) of the ribosomal DNA (rDNA) was sequenced from all isolates to determine genetic identity. A single anastomosis group (AG) of binucleate Rhizoctonia represented 92% of the samples collected from infected leaves. Genetic data and hyphal fusion experiments confirmed that these isolates belong to AG-U, which was recently identified from root and stem infections on miniature rose in Japan. Isolates of binucleate Rhizoctonia belonging to anastomosis groups AG-R, CAG-7 (=AG-S), and AG-G were also identified in the sample in low frequency. This is the first report of the occurrence of binucleate Rhizoctonia AG-U in the United States.

Infection and Fumonisin Production by Fusarium verticillioides in Developing Maize Kernels.

ABSTRACT Fusarium ear rot and fumonisin contamination are serious problems for maize growers, particularly in the southeastern United States. The lack of maize genotypes highly resistant to infection by Fusarium verticillioides or to fumonisin contamination emphasizes the need for management strategies to prevent contamination by this mycotoxin. Information on the initial appearance of infection and fumonisin contamination of kernels and their increase over time is needed to determine if early harvest may be an appropriate control strategy. Maize ears from replicated studies at two locations in eastern North Carolina were harvested weekly, starting 2 weeks after pollination and continuing for 14 weeks. The percentage of kernels infected with F. verticillioides and the fumonisin contamination in the harvested samples were determined. Kernel infection by F. verticillioides and fumonisin contamination appeared as kernels neared physiological maturity and increased up to the average harvest date for maize in North Carolina. Beyond this date, the concentrations of fumonisin fluctuated. Under years conducive for fumonisin contamination, early harvest (greater than 25% grain moisture) may help reduce the level of contamination.

Weed Species as Hosts of Sclerotinia minor in Peanut Fields.

Bleached stems and sclerotia were observed on winter annual weed species growing in harvested peanut fields in northeastern North Carolina in March 2001. Each field had a history of Sclerotinia blight caused by Sclerotinia minor. Symptomatic plants were collected and brought back to the laboratory for identification and isolation. S. minor was isolated and Koch's postulates were fulfilled to confirm pathogenicity of S. minor on nine weed species. They included Lamium aplexicaule (henbit), Cardamine parviflora (smallflowered bittercress), Stellaria media (common chickweed), Cerastium vulgatum (mouse-ear chickweed), Coronopus didymus (swinecress), Oenothera laciniata (cutleaf eveningprimrose), Conyza canadensis (horseweed), Brassica kaber (wild mustard), and Arabidopsis thaliana (mouse-ear cress). This is the first report of these species as hosts of S. minor in the natural environment. All isolates of S. minor obtained from the weed species were pathogenic to peanut.

Phytophthora infestans Populations from Tomato and Potato in North Carolina Differ in Genetic Diversity and Structure.

ABSTRACT Phytophthora infestans causes a destructive disease on tomato and potato. In North Carolina (NC) potatoes are mostly grown in the east, whereas tomatoes are grown in the mountainous areas in the western part of the state. Five genotypes of P. infestans were identified from 93 and 157 isolates collected from tomato and potato over a 5 year period between 1993 and 1998. All isolates collected from potato in eastern NC were the US-8 genotype, whereas only a single isolate was the US-1 genotype. Tuber blight was found on immature daughter tubers in a single field in 1997, however infection on mature tubers was not observed. Within potato fields, a range of sensitivity to metalaxyl was observed among isolates but all were either intermediate or highly resistant to the fungicide. In contrast, isolates from tomatoes included previously reported US-7 and US-8 genotypes and two new genotypes called US-18 and US-19 (A2 mating type, allozyme genotype Gpi 100/100 and Pep 92/100). These genotypes had unique restriction fragment length polymorphism banding patterns, were sensitive to metalaxyl, and have not been reported elsewhere. All genotypes, with the exception of the US-1, were the Ia mitochondrial haplotype. Thus, isolates of P. infestans from tomato were more genetically diverse over time in NC than those from potato and include two new genotypes that are sensitive to metalaxyl.

Viability and stability of biological control agents on cotton and snap bean seeds.

Cotton and snap bean were selected for a multi-year, multi-state regional (south-eastern USA) research project to evaluate the efficacy of both commercial and experimental bacterial and fungal biological control agents for the management of damping-off diseases. The goal for this portion of the project was to determine the viability and stability of biological agents after application to seed. The biological seed treatments used included: (1) Bacillaceae bacteria, (2) non-Bacillaceae bacteria, (3) the fungus Trichoderma and (4) the fungus Beauveria bassiana. Seed assays were conducted to evaluate the following application factors: short-term (< or = 3 months) stability after seed treatment; quality (i.e. isolate purity); compatibility with chemical pesticides and other biocontrol agents; application uniformity between years and plant species. For the bacterial treatments, the Bacillaceae genera (Bacillus and Paenibacillus) maintained the greatest population of bacteria per seed, the best viability over time and the best application uniformity across years and seed type. The non-Bacillaceae genera Burkholderia and Pseudomonas had the least viability and uniformity. Although Beauveria bassiana was only evaluated one year, the seed fungal populations were high and uniform. The seed fungal populations and uniformity for the Trichoderma isolates were more variable, except for the commercial product T-22. However, this product was contaminated with a Streptomyces isolate in both the years that it was evaluated. The study demonstrated that Bacillaceae can be mixed with Trichoderma isolates or with numerous pesticides to provide an integrated pest control/growth enhancement package.

Evaluation of Wounds as a Factor to Infection of Cabbage by Ascospores of Sclerotinia sclerotiorum.

A semi-selective medium was used to examine the aerobiology of ascospores of Sclerotinia sclerotiorum in five commercial cabbage fields in eastern North Carolina. Ascospores were present in all five fields from 26 September to 30 November. However, numbers of ascospores varied greatly depending on location, sampling date, and time. In general, peak ascospore deposition occurred between 11:00 A.M. and 1:00 P.M., with the number of colonies recovered ranging from 3 to 55/dish (9 cm in diameter). Peak ascospore numbers at all locations were found from mid- to late October, but a second, smaller peak was also evident at each location in late November. Information obtained was employed to evaluate the role of wounding in infection of cabbage by ascospores of S. sclerotiorum in controlled environmental chambers. A method for production and release of ascospores of S. sclerotiorum was employed in controlled-environment chambers for the inoculation of cabbage plants with one of three representative foliar wounds: a bruise, a cut, or a non-lethal freeze. Wounding treatments were applied to 7-week-old cabbage plants, misting was added to maintain continuous leaf wetness, and ascospores were released from apothecia twice daily for four consecutive days. Spore trapping with a semi-selective medium indicated that inoculum was evenly distributed within the chambers and deposition was similar to levels recorded in the field. At 31 days after inoculation, disease incidence ranged from 0% on the control to 96% on the freeze treatments. Freeze-treated plants showed the highest disease severity throughout the entire incubation period. Mean area under the disease progress curve of severity values were 0, 0.2, 34 and 60 for the control, cut, bruise, and freeze treatments, respectively. Results indicate that freeze and bruise injuries are important factors associated with infection of cabbage by S. sclerotiorum.

First Report of Plasmodiophora brassicae on Cabbage in Eastern North Carolina.

Clubroot, caused by Plasmodiophora brassicae Woronin, has occurred for at least 50 years in three counties in northwestern North Carolina, but has not been reported previously from eastern North Carolina, where most commercial cabbage is produced. In the fall of 1995, clubroot was observed in a direct seeded, commercial cabbage field in Plymouth, NC. Diseased cabbage plants were stunted and roots exhibited clublike swellings. Clubs were randomly harvested from roots of five plants to obtain a composite isolate to determine which race(s) of P. brassicae are infecting cabbage in eastern North Carolina. Three experiments were conducted, using the procedure of Williams (2). Four replicates of 10, 1-week-old seedlings of eight different crucifer cultivars were inoculated by dipping in a spore suspension (1 × 108 cysts/ml) of P. brassicae and planted in pasteurized potting mix. Seedlings dipped in sterile water served as controls. Inoculated seedlings were incubated in a greenhouse at 18 to 28°C for 6 to 8 weeks and assessed for clubroot incidence and severity. The isolate of P. brassicae from eastern North Carolina was most virulent on cabbage (Brassica oleracea var. capitata cv. Jersey Queen), collard (B. oleracea var. acephala cv. Vates), and wild mustard (B. nigra); moderately virulent on canola (B. napus cv. Brutor) and rutabaga (B. napus cvs. Laurentian and Wilhelmsburger); and least virulent on cabbage (cv. Badger Shipper). Canola (B. napus cv. Nevin) and control seedlings were not infected and exhibited no symptoms. Similar results were obtained for all experiments. Based on these results, the isolate of P. brassicae from eastern North Carolina was designated as race 6 and pathotype 5 according to Williams (2) and Some (1), respectively. However, further experiments with single-cyst-derived isolates from individual clubs obtained from different geographic locations are needed to accurately characterize field populations of P. brassicae on cabbage in eastern North Carolina. References: (1) A. Some et al. Plant Pathol. 45:432, 1996. (2) P. H. Williams. Phytopathology 56:624, 1966.

Mushroom poisoning.

The majority of cases of mushroom poisoning occur in children and involve benign gastrointestinal irritants. Critical poisonings most frequently occur in adults who ingest Amanita phalloides or other mushrooms containing amanitin. Critical versus noncritical poisonings can be diagnosed with a high degree of confidence by the patient's history and initial symptoms. The most promising new medical treatment for Amanita mushroom poisoning is silibinin. In suspected cases of mushroom poisoning, it is important to obtain specimens of the ingested mushrooms, if possible, since treatment is specific to the species.

Clonality in Sclerotinia sclerotiorum on Infected Cabbage in Eastern North Carolina.

ABSTRACT Eighty-four isolates of Sclerotinia sclerotiorum from four cabbage production fields in North Carolina and 16 isolates from an experimental cabbage field plot in Louisiana were DNA-fingerprinted and tested for mycelial compatibility. In a comparison with 594 unique DNA fingerprints of S. sclerotiorum from Canadian canola, no fingerprints were shared among Canadian, North Carolina, and Louisiana populations. DNA fingerprints from the North Carolina sample were distinctive from those of the Canadian and Louisiana samples, with significantly more hybridizing fragments in the 7.7- to 18-kilobase range. Forty-one mycelial compatibility groups (MCGs) and 50 unique DNA fingerprints were identified from the North Carolina sample. Three MCGs and three fingerprints were identified from the Louisiana sample. From the North Carolina sample, 32 MCGs were each associated with a unique fingerprint; of these, there were 11 clones (i.e., cases in which two or more isolates belonged to the same MCG and shared the same DNA fingerprint). Six clones sampled from two or more fields represented approximately 29% of the total sample (24 of 84 isolates), with six clones recovered from fields 75 km apart. There were 10 cases in which one MCG was associated with more than one DNA fingerprint and two cases in which one DNA fingerprint was associated with more than one MCG. The small sample from Louisiana was strictly clonal. The North Carolina sample had a clonal component, but deviated from one-to-one association of MCG with DNA fingerprint to an extent consistent with more recombination or transposition than the other two populations sampled.