Marc A. Cubeta

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.

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.

Genetic diversity of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina

Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

BackgroundThe soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).ResultsPopulations of R. solani AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of R. solani AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of R. solani AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (ΦST= 0.257, significant at P < 0.05) but not for locus pP42F (ΦST= 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of R. solani AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of R. solani AG-3.ConclusionThe two sisters populations of the basidiomycete fungus R. solani AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.

Genetic structure of populations of Rhizoctonia solani AG-3 on potato in eastern North Carolina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solani anastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 PT on potato that includes both recombination and clonality.

Biological control of rhizoctonia and pythium damping-off of cucumber: An integrated approach

Abstract Binucleate Rhizoctonia spp. were evaluated singly and in combination with seed treated with selected biocontrol bacteria, fungicides, or osmopriming agents in the greenhouse and field for control of Rhizoctonia and Pythium damping-off of cucumber. In the greenhouse, in a pasteurized soil mix, treatments with 8 of 19 isolates of binucleate Rhizoctonia spp. yielded decreased (P = 0.05) levels of preemergence damping-off caused by R. solani. Osmopriming cucumber seed with KNO3, NaCl, or polyethylene glycol (PEG 8000) did not protect cucumber from Rhizoctonia and Pythium damping-off in naturally infested soil in the greenhouse. Treating cucumber seed with strain E6, ECH, and ECT of Enterobacter cloacae and sowing in soil alone or in soil amended with isolate 232-CG of binucleate Rhizoctonia sp. resulted in decreased (P = 0.05) levels of Rhizoctonia and Pythium preemergence damping-off in naturally infested soil in the greenhouse but not in the field. Amendment of isolate 232-CG to soil and combining it with seed treatment or soil application of metalaxyl produced decreased (P = 0.05) levels of Rhizoctonia and Pythium preemergence damping-off in 12 naturally infested fields at three geographic locations when compared with nontreated seed sown in soil alone or in soil amended with isolate 232-CG alone. Seed treated with metalaxyl and sown in soil amended with isolate 232-CG developed decreased (P = 0.05) levels of Rhizoctonia and Pythium preemergence damping-off at 20 and 30% of the field sites compared to seed treated with captan and metalaxyl, respectively. However, seed treated with metalaxyl or captan had higher (P = 0.05) disease incidence of Rhizoctonia at 20 and 60% of the field sites, respectively, when compared to seed treated with metalaxyl and sown in soil amended with isolate 232-CG. Control of cucumber damping-off caused by R. solani and Pythium spp. requires an integrated approach and can be achieved using the binucleate Rhizoctonia sp. isolate 232-CG combined with the fungicide metalaxyl. This treatment protected cucumber from Rhizoctonia and Pythium damping-off as effectively as the recommended fungicide captan.

Mobile elements and mitochondrial genome expansion in the soil fungus and potato pathogen Rhizoctonia solani AG-3

The soil fungus Rhizoctonia solani is an economically important pathogen of agricultural and forestry crops. Here, we present the complete sequence and analysis of the mitochondrial genome of R. solani, field isolate Rhs1AP. The genome (235 849 bp) is the largest mitochondrial genome of a filamentous fungus sequenced to date and exhibits a rich accumulation of introns, novel repeat sequences, homing endonuclease genes, and hypothetical genes. Stable secondary structures exhibited by repeat sequences suggest that they comprise functional, possibly catalytic RNA elements. RNA-Seq expression profiling confirmed that the majority of homing endonuclease genes and hypothetical genes are transcriptionally active. Comparative analysis suggests that the mitochondrial genome of R. solani is an example of a dynamic history of expansion in filamentous fungi.

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.

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.

Phylogenetic relatedness of the M2 double-stranded RNA in Rhizoctonia fungi

Isolates from closely related fungi in the Rhizoctonia species complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia (AGA, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3′ region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus (anamorph = R. solani AG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidium that also is present in Thanatephorus.