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Since 2006 there has been a decline in Colorado blue spruce (CBS; Picea pungens ) planted as landscape trees and for Christmas tree production throughout the Lower Peninsula of Michigan. This decline is characterized by a slow loss of needles in the lower portion of the tree starting at branch tips, followed by entire branch dieback, which progresses upward over several years. This dieback has been linked to shallow branch cankers visible in the phloem when the bark layer is removed. Isolates in the fungal genus Diaporthe have been consistently isolated from lesion margins on symptomatic branches. Before the initial reports of declining CBS in landscape and Christmas trees, Diaporthe was known only as a nursery disease of CBS. To determine the species of Diaporthe linked to the decline of CBS in Michigan, seven gene regions were sequenced from a collection of Diaporthe isolates collected in 2011 through 2018 from CBS and other coniferous hosts. Subsequent phylogenetic analyses indicated that Diaporthe eres and a novel Diaporthe clade were present on symptomatic CBS in Michigan. The new species D. brevicancria nov. is described, and Koch's postulates were confirmed for D. brevicancria nov. and D. eres . D. brevicancria nov. produced the largest cankers in greenhouse pathogenicity trials, and dual inoculations of D. brevicancria nov. and D. eres produced intermediate cankers.

In the early 2000s, spruce trees in Michigan began displaying basal needle drop and branch death that slowly progressed upward, symptoms of what we call spruce decline. A survey in 2013 revealed that spruce decline was common throughout Michigan's Lower Peninsula, and Diaporthe was the most likely pathogen causing the cankers associated with these symptoms. Greenhouse inoculation studies completed Koch's postulates, confirming that Diaporthe could cause cankers that cause needle loss and branch death. The five different Diaporthe haplotypes isolated from symptomatic branches during the survey differed in virulence. Haplotypes 2, 4, and 5 were more virulent, and differed from each other by only one or two base pairs using the internal transcribed spacer (ITS) region and did not differ using the β-tubulin (TUB) gene. These haplotypes were unresolved phylogenetically. Haplotypes 1 and 3 were weakly virulent to avirulent on multiple spruce taxa, and fell into a resolved Diaporthe eres clade. Spruce taxa varied in susceptibility, with Colorado blue spruce (Picea pungens) the most susceptible, followed by Norway (P. abies), then white spruce (P. glauca). Spruce taxa that were much less susceptible were Black Hills (P. glauca var. densata), Serbian (P. omorika), and Meyer spruce (P. meyeri). We demonstrate that one or more Diaporthe species is causing cankers on declining spruce in Michigan, and these cankers elicit symptoms similar to the branch death expressed by declining spruce in the landscape. Future work will focus on further characterizing Diaporthe to species, and determining biotic and abiotic stressors that may predispose spruce trees to express decline symptoms.

Chestnut blight cankers, caused by the fungus Cryphonectria parasitica, are prone to invasion by other microorganisms as the canker ages. This microbial community has the potential to alter canker expansion, which may influence the probability that the canker girdles the infected stem. Hypoviruses infect the pathogen mycelium directly and are known to decrease pathogen virulence (i.e. hypovirulent). These viral infections can slow pathogen growth, decreasing the rate of canker expansion and lowering the probability of girdling. Saprophytic fungi also invade the expanding canker and may antagonize C. parasitica leading to reduced pathogen growth. The combined effects of fungal antagonism and a hypovirulent pathogen could work in combination to reduce the probability of girdling the infected stem. We assessed the ability of different fungal taxa, isolated from low severity cankers, to inhibit the growth of virulent and hypovirulent forms of C. parasitica in dual culture tests on two cultural media. Percent growth inhibition of virulent C. parasitica by potentially antagonistic fungi ranged from 2 % to 34 %, while inhibition of hypovirulent C. parasitica ranged from 18 % to 54 %. Only one isolate, identified as Umbelopsis isabellina (UmbelopsisWS) inhibited the virulent form of the pathogen more than the hypovirulent form. All three Trichoderma isolates caused the greatest growth inhibition of virulent C. parasitica, but they, like all other fungal isolates tested, inhibited the hypovirulent form of the pathogen more than the virulent form. These results suggest that commonly occurring fungi in chestnut blight cankers, including Trichoderma, may inhibit the hypovirulent C. parasitica more than virulent C. parasitica. Thus, the presence of other fungi in cankers may not enhance the effect of hypovirulent C. parasitica to delay cankers from girdling a stem but instead intensify canker development., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Hypovirus-infected Cryphonectria parasitica strains were introduced in a large stand of American chestnut (>4,000 individuals) in western Wisconsin (USA) to evaluate whether hypoviruses can serve as biological control agents. They were deployed by treating cankers from 1992 to 1997 and again from 2004 to 2014. After 17 years of hypovirus introductions within an area of the stand with the longest history of disease, isolation of hypovirus-infected strains increased from 55% in 1994 to 86% in 2014 from cankers that were treated. During the same period, isolation from cankers that arose on trees with treated cankers increased from 29 to 72% and from 15 to 84% for cankers on nearby trees that received no treatment. Tree survivorship over the 23-year study period for trees with treated cankers was 51% compared with 31% for trees that were not treated. Introduction of hypovirus has resulted in the regrowth of the crowns of many large-diameter trees. Putative recovery of American chestnut in this stand provides evidence that prolonged hypovirus treatment can act as a biological control when limited numbers of vegetative compatibility types of C. parasitica exist.

Background: Chestnut is a relatively new cultivated crop for Michigan, and postharvest loss due to decay has been problematic as production has increased each year. In 2007, more than 25% of chestnuts were lost to postharvest decay, equivalent to approximately 5300 kg of fresh product. To determine the organisms responsible for decay, a microbiological survey was performed in 2006 and 2007 to identify microorganisms involved in postharvest shell (external surface) mold and internal kernel (edible portion) decay of chestnuts., Results: Filamentous fungi including Penicillium expansum, Penicillium griseofulvum, Penicillium chrysogenum, Coniophora puteana, Acrospeira mirabilis, Botryosphaeria ribis, Sclerotinia sclerotiorum, Botryotinia fuckeliana (anamorph Botrytis cinerea) and Gibberella sp. (anamorph Fusarium sp.) were the predominant microorganisms that negatively impacted fresh chestnuts. Populations of microorganisms varied between farms, harvesting methods and chestnut parts., Conclusion: Chestnuts harvested from the orchard floor were significantly (P < 0.05) more contaminated than chestnuts harvested directly from the tree, by more than 2 log colony-forming units (CFU) g(-1) . In addition, a significant difference (P < 0.05) in the microbial population was seen between chestnuts submitted by different growers, with average count ranges of fungi, mesophilic aerobic bacteria (MAB) and yeasts equal to 4.75, 4.59 and 4.75 log CFU g(-1) respectively. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Vegetative compatibility (VC) is commonly used to characterize structure and diversity in fungal populations. In the chestnut blight fungus, Cryphonectria parasitica, high VC diversity is hypothesized to be responsible for the failure of hyperparasitic mycoviruses to spread through pathogen populations in North America. To test this hypothesis, we assessed VC diversity at three recovering sites in Michigan where mycoviruses had invaded and compared them with four epidemic population sites where mycoviruses were absent. VC diversity was assessed for samples collected in 1996 and 2009, which allowed us to determine how C. parasitica populations changed with time. Twelve VC types were found in 1996 while 29 were found in 2009; 75% of types overlapped between the sample dates. Sites where mycoviruses were present had unique VC structures with the exception of the recovering population site at County Line where the main VC group was also detected at two epidemic sites. With one exception, epidemic sites contained more VC groups and displayed higher population level diversity than recovering sites. Mating-type analyses of blight populations revealed that two of three recovering populations were significantly skewed for MAT2 suggesting asexual reproduction, while epidemic sites with a long history of blight infection had ratios near 50:50 suggesting sexual reproduction. We propose that selection in the largely asexual C. parasitica populations at two recovering sites favors the most-fit fungal genotype by mycovirus combination and results in reduced diversity relative to the sexually reproducing pathogen populations at epidemic sites.

Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato, is distinguished into four fingerprint types (A, B, C, and D) using BOX-polymerase chain reaction (PCR). To characterize the variation within the C. michiganensis subsp. michiganensis population in Michigan, 718 strains of C. michiganensis subsp. michiganensis were isolated from infected foliage and fruit collected from 14 and 9 Michigan commercial tomato fields in 1997 and 1998, respectively. The frequency of PCR types detected with BOX-PCR in all strains, and Bayesian cluster analysis, pairwise differentiation index comparisons, and genetic diversity estimates of 96 strains genotyped for six virulence-related genes revealed that C. michiganensis subsp. michiganensis populations in Michigan tomato fields are geographically structured. A multilocus haplotype cladogram was also consistent with geographic stratification in C. michiganensis subsp. Michiganensis populations. Some regions had strains predominantly of only one PCR type or belonging mostly to one genetic cluster, while other regions presented more diversity of occurrence of PCR types and genetic clusters. Results derived from this study provide information about the genetic structure of C. michiganensis subsp. michiganensis populations in Michigan and genetic diversity of C. michiganensis subsp. michiganensis inocula, which is key in developing disease management strategies.

Chestnut extracts were studied for antimicrobial activity against selected microorganisms, including plant pathogens. Chestnut extract on paper discs was applied to an agar medium to evaluate the inhibition to multiple microorganisms or the extract was added at various concentrations to a culture medium to evaluate the growth of target microorganisms. Chestnut type, tissue of plants (shell, pellicle, and leaf), extraction methods, and physical characteristics were studied to determine antimicrobial activity. Most test microorganisms were inhibited by the extracts at different effective concentrations for 50% growth inhibition (EC 50 ). Pseudomonas fluorescens was the most sensitive (EC 50 = 4.4 μg/μl), Phytophthora cambivora was one of the least inhibited (EC 50 = 185 μg/μl), and Cryphonectria parasitica was not inhibited. Extracts of the Japanese × European chestnut (Castanea crenata × C. sativa) 'Colossal' showed a greater inhibition than those of wild trees of the Chinese species (C. mollissima). High temperature did not affect the inhibitory effect. Extracts from chestnut pellicle had the highest concentration of antimicrobial compound, compared with leaf and shell. The active fraction contained several substances with molecular masses consistent with one flavonol glycoside and several terpenoid substances. Pellicle and shell tissue reduced radish scab disease caused by Streptomyces scabies in the greenhouse.

In the chestnut blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes are elicited by debilitating mitochondrial DNA (mtDNA) mutations. In virus-free hypovirulent strains of C. parasitica from nature, the presence of a mitochondrial DNA element, named InC9, has been reported to cause similar disease syndromes. We have detected an additional mitochondrial element, termed plME-C9 (plasmid-like mitochondrial element C9) in some of the strains rendered hypovirulent by InC9. This element is a 1.4-kb DNA that exists in the mitochondria as monomeric and multimeric circular forms. Only a short 127-bp sequence of the plME-C9 DNA is derived from a region of the C. parasitica mtDNA that contains a reverse transcriptase-like open reading frame. The accumulation of the plME-C9 DNA in the mitochondria appears to adversely affect the growth of the fungus on synthetic medium. However, the presence plME-C9 in different strains did not correlate with the manifestation of the hypovirulence phenotype, indicating that it is not the primary reason for the prevalence of attenuated C. parasitica strains in the Kellogg Forest in Michigan., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the chestnut-blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes frequently are elicited by double-stranded RNA (dsRNA) virus infections. However, some strains manifest cytoplasmically transmissible hypovirulence traits without containing any mycovirus. In this study, we describe an altered form of mtDNA that is associated with hypovirulence and senescence in a virus-free strain of C. parasitica, KFC9, which was obtained from nature and has an elevated level of cyanide-resistant respiration. In this strain, a 971-bp DNA element, named InC9, has been inserted into the first exon of the mitochondrial small-subunit ribosomal RNA (rns) gene. Sequence analysis indicates that InC9 is a type A1 group II intron that lacks a maturase-encoding ORF. RT-PCR analyses showed that the InC9 sequence is spliced inefficiently from the rRNA precursor. The KFC9 strain had very low amounts of mitochondrial ribosomes relative to virulent strains, thus most likely is deficient in mitochondrial protein synthesis and lacks at least some of the components of the cyanide-sensitive, cytochrome-mediated respiratory pathway. The attenuated-virulence trait and the splicing-defective intron are transferred asexually and concordantly by hyphal contact from hypovirulent donor strains to virulent recipients, confirming that InC9 causes hypovirulence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

The mt-rns gene of Cryphonectria parasitica is 9872bp long and includes two group I and two group II introns. An analysis of intronic protein-encoding sequences revealed that LAGLIDADG ORFs, which usually are associated with group I introns, were transferred at least twice into group II introns. A plasmid-like mitochondrial element (plME) that appears in high amounts in previously mutagen-induced mit1 and mit2 hypovirulent mutants of the Ep155 standard virulent strain of C. parasitica was found to be derived from a short region of the mt-rns gene, including the exon 1 and most of the first intron. The plME is a 4.2-kb circular, multimeric DNA and an autonomously-replicating mtDNA fragment. Although sexual transmission experiments indicate that the plME does not directly cause hypovirulence, its emergence is one manifestation of the many complex molecular and genetic events that appear to underlie this phenotype.

Phytophthora cinnamomi, P. drechsleri, P. citricola, and P. cactorum limit Fraser fir production, whereas P. capsici affects Solanaceous, Cucurbitaceous, and Fabaceous crops. Some vegetable growers in Michigan plant conifers for the Christmas tree market in fields infested with P. capsici. To determine the susceptibility of Fraser fir to P. capsici, stems (no wound or 1- or 3-mm-diameter wound) or roots (2 or 4 g of infested millet seed or 2 or 5 × 10 3 zoospores/ml of a zoospore suspension) of seedlings were inoculated with each of four P. capsici isolates and incubated in growth chambers (20 or 25°C). In addition, Fraser fir seedlings were planted in two commercial fields naturally infested with P. capsici. All P. capsici isolates tested incited disease in the seedlings regardless of incubation temperature or inoculation method. Seedlings (72%) planted in P. capsici-infested fields developed disease symptoms and died. Most of the P. capsici isolates obtained from the Fraser fir seedlings infected while in the field were recovered from root tissue. Identification was confirmed by species-specific direct colony polymerase chain reaction. The pathogen was successfully recovered from stems of all stem-inoculated seedlings, and from roots and stems of all root-inoculated seedlings; the phenotype of the recovered isolate matched the phenotype of the inoculum. This study suggests that planting Fraser fir in fields infested with P. capsici could result in infection and that adjustments in current rotational schemes are needed.

Symptomless greenhouse tomato transplants may harbor high populations of Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker, leading to yield loss in the field. The objective of this study was to determine whether resistant cultivars, acibenzolar-S-methyl, avirulent strains of C. michiganensis subsp. michiganensis, or standard bactericides reduce pathogen populations and spread among greenhouse tomato seedlings. All treatments limited pathogen populations compared with the untreated inoculated susceptible cultivar in 1996 and 1998, but not in 1997. In 1996, copper hydroxide alone or mixed with mancozeb or streptomycin limited pathogen populations relative to acibenzolar-S-methyl, acibenzolar-S-methyl mixed with copper hydroxide, and avirulent strains. Copper hydroxide mixed with streptomycin limited pathogen populations compared with copper hydroxide mixed with mancozeb. Adding copper hydroxide to acibenzolar-S-methyl limited pathogen populations compared with acibenzolar-S-methyl alone. In 1998, treatments did not differ significantly from each other in limiting pathogen populations. The treatments limited spread of the bacterium only in 1997. Copper hydroxide mixed with mancozeb limited spread compared with copper hydroxide mixed with streptomycin. Pathogen spread was also reduced among resistant cultivars compared with the susceptible cultivar treated with streptomycin. In the field, the untreated inoculated susceptible cultivar produced yields that were 61% (1996) and 93% (1997) of those produced by the uninoculated susceptible cultivar. Fruit spotting occurred regardless of treatment. To prevent severe bacterial canker disease in the field, growers should initiate and sustain bactericide applications to tomato transplants while in the greenhouse to suppress pathogen populations. Cultivar resistance and acibenzolar-S-methyl may be helpful in disease management of bacterial canker on tomato.

Development of the bird's eye fruit lesion of tomato was studied by inoculating flowers and the surface of young tomato fruit with strains of Clavibacter michiganensis subsp. michiganensis. Flowers were sprayed once or twice with C. michiganensis subsp. michiganensis at 10 8 CFU/ml. The maximum incidence (80%) and severity (12 spots/fruit) of spotted fruit resulted when inoculum was sprayed twice, 3 days apart. Flowers were most susceptible to infection 2 days after anthesis. When a paintbrush was used to apply inoculum to the surface of small fruit, a large number of fruit spots (≤456 spots/fruit) resulted. Even strains determined to be avirulent based on a tomato stem inoculation assay and a hypersensitive response on four-o'clock leaves (Mirabilis jalapa) were able to produce fruit spots, although at a reduced level. The inoculation methods developed in this study can provide opportunities to observe subtle host-pathogen interactions between C. michiganensis subsp. michiganensis strains and tomato and to help formulate methods to quantify infection.

In the chestnut-blight fungus Cryphonectria parasitica, a plasmid, pCRY1, occurs in the mitochondria of several strains isolated at various locations in the northeastern United States and Canada. The monomer of this plasmid is a 4.2-kb circular double-stranded DNA that has no detectable sequence homology with the 160-kb mitochondrial DNA of Ep155, a standard virulent laboratory strain of C. parasitica. The circular nature and oligomeric characteristics of the plasmid were deduced from the heterogeneous size of plasmid DNA molecules as detected by one- and two-dimensional gel-electrophoresis, the nature and alignment of restriction fragments, and the lack of detectable termini in the nucleotide sequence. The cytoplasmic location of the plasmid was deduced from its co-purification with mitochondria, uniparental (maternal) transmission in sexual crosses, dissociation from the nuclei of the donor strain during its horizontal transfer between vegetatively compatible strains through hyphal anastomoses, and mitochondrial codon usage (UGA = Try). The pCRY1 plasmid contains a long open reading frame that is transcribed and potentially encodes a unique 1214 amino-acid, B-family DNA polymerase similar to those encoded by the LaBelle and Fiji circular mitochondrial plasmids of Neurospora. In this subgroup of proteins, the DTD motif characteristic of B-family DNA polymerases is replaced by TTD. Amino-acid motifs related to those that are characteristic of the 3'-->5' exonuclease domains of B-family DNA polymerases have been located in the amino-terminal portion of the proteins. A comparison of isogenic plasmid-free and plasmid-containing cultures indicates that pCRY1 is an infectious agent that effects a reduction in the pathogenicity of some, but not all, strains of C. parasitica.

ABSTRACT Chemical applications, with the exception of mancozeb, reduced population sizes and spread of Clavibacter michiganensis subsp. michiganensis among tomato seedlings in the greenhouse and impacted subsequent plant development and yield in the field. While applications of copper hydroxide, copper hydroxide/mancozeb, copper hydroxide/mancozeb (premixed 12 h before spraying), streptomycin, and streptomycin/copper hydroxide to seedlings in the greenhouse did not differ significantly from the inoculated control, the trend was for these treatments to increase the survival of inoculated transplants in the field in comparison to the inoculated control. In the field, inoculated controls produced yields that were 63% (1995) and 51% (1996) of those produced by uninoculated controls. In both years, with the exception of mancozeb in 1995, all treatments resulted in yields similar to those obtained with the uninoculated control. Plant survival and yield in the field were severely affected when transplants had a pathogen population of >/= x 10(8) CFU/g of tissue. All treatments, with the exception of mancozeb, limited C. michiganensis subsp. michiganensis populations to <5.0 x 10(5). None of the treatments significantly reduced the incidence of fruit spotting compared with that of the inoculated control.

A cytoplasmically transmissible hypovirulence syndrome has been identified in virus-free strains of the chestnut blight fungus Cryphonectria parasitica isolated from healing cankers on American chestnut trees in southwestern Michigan. The syndrome is associated with symptoms of fungal senescence, including a progressive decline in the growth potential and abundance of conidia, and elevated levels of respiration through the cyanide-insensitive alternative oxidase pathway. Conidia from senescing mycelia exhibited varying degrees of senescence ranging from normal growth to death soon after germination. Cytoplasmic transmission of hypovirulence between mycelia occurred by hyphal contact and coincided with the transfer of a specific restriction fragment length polymorphism from the mitochondrial DNA (mtDNA) of the donor strains into the mtDNA of virulent recipients. The transmission of the senescence phenotype was observed not only among vegetatively compatible strains but also among incompatible strains. Hypovirulence was present in isolates from the same location with different nuclear genotypes as identified by DNA fingerprinting. This study confirms that mitochondrial hypovirulence can occur spontaneously and spread within a natural population of a phytopathogenic fungus.

Isolate Grand Haven (GH) 2 is a naturally occurring isolate of the chestnut blight fungus, Cryphonectria parasitica, that is greatly reduced in virulence due to the presence of a double-stranded RNA virus. Unlike many other virus-infected, hypovirulent isolates, GH2 is not substantially reduced in pigmentation, conidiation, or laccase expression compared to its virus-free counterpart. The dsRNA genome of the GH2 virus was cloned, sequenced, and compared to hypovirulence-associated viruses of the family Hypoviridae. GH2 dsRNA is considerably smaller than previously characterized members of the family, 9.8 kb compared to 12.5-12.7 kb for other members. The genome organization of GH2 dsRNA reflected the substantial difference in genome size. Like other members of the family, one strand contained a poly(A)(+) tail at the 3' end and a long sequence with several minicistrons at the 5' end of the same strand. Only a single open reading frame (ORF) of 8622 nucleotides was predicted from deduced translations of the poly(A)(+)-containing strand, however. This contrasts with the two-ORF structures of previously characterized members. Analysis of the deduced ORF of GH2 dsRNA revealed putative proteinase, RNA polymerase, and helicase domains similar to those previously identified in confirmed members of the virus family Hypoviridae. GH2 dsRNA was more distantly related to Cryphonectria hypovirus (CHV) 1-EP713 and CHV2-NB58 than the latter two were to each other but has features in common with each of those viruses. We propose that the GH2 virus be included in this taxon as a member of the genus Hypovirus, representing a strain of a new species, CHV3., (Copyright 1999 Academic Press.)

A 4238-bp intervening sequence within the highly conserved U11 region of the mitochondrial large subunit ribosomal RNA gene of the fungus Cryphonectria parasitica Ep155 has been sequenced and identified to be a group-I intron. This is the largest group-I intron reported to-date for fungal mitochondrial genomes. The intron contains an 851-codon open reading frame encoding a putative, but complete, small-subunit ribosomal protein of 510 amino acids which is fused at its carboxyl terminus to a 311 amino-acid polypeptide representing a typical maturase-like protein. A short open reading frame of 83 amino acids with some similarity to maturases, but lacking a translation-initiation codon, was also noted at the 3' end of the intron. The unusual size of the intron and the arrangement of the open and truncated reading frames suggest that this segment of the mtDNA of C. parasitica has arisen by a fusion of components from two or more different introns, possibly involving the re-location of intronic genes.

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.

A major focus of research on the dynamics of host-pathogen interactions has been the evolution of pathogen virulence, which is defined as the loss in host fitness due to infection. It is usually assumed that changes in pathogen virulence are the result of selection to increase pathogen fitness. However, in some cases, pathogens have acquired hypovirulence by themselves becoming infected with hyperparasites. For example, the chestnut blight fungus Cryphonectria parasitica has become hypovirulent in some areas by acquiring a double-stranded RNA hyperparasite that debilitates the pathogen, thereby reducing its virulence to the host. In this article, we develop and analyze a mathematical model of the dynamics of host-pathogen interactions with three trophic levels. The system may be dominated by either uninfected (virulent) or hyperparasitized (hypovirulent) pathogens, or by a mixture of the two. Hypovirulence may allow some recovery of the host population, but it can also harm the host population if the hyperparasite moves the transmission rate of the pathogen closer to its evolutionarily stable strategy. In the latter case, the hyperparasite is effectively a mutualist of the pathogen. Selection among hyperparasites will often minimize the deleterious effects, or maximize the beneficial effects, of the hyperparasite on the pathogen. Increasing the frequency of multiple infections of the same host individual promotes the acquisition of hypovirulence by increasing the opportunity for horizontal transmission of the hyperparasite. This effect opposes the usual theoretical expectation that multiple infections promote the evolution of more virulent pathogens via selection for rapid growth within hosts.

In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155.

Mutations causing mitochondrial defects were induced in a virulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. Virulence on apples and chestnut trees was reduced in four of six extensively characterized mutants. Relative to the virulent progenitor, the attenuated mutants had reduced growth rates, abnormal colony morphologies, and few asexual spores, and they resembled virus-infected strains. The respiratory defects and attenuated virulence phenotypes (hypovirulence) were transmitted from two mutants to a virulent strain by hyphal contact. The infectious transmission of hypovirulence occurred independently of the transfer of nuclei, did not involve a virus, and dynamically reflects fungal diseases caused by mitochondrial mutations. In these mutants, mitochondrial mutations are further implicated in generation of the attenuated state by (i) uniparental (maternal) inheritance of the trait, (ii) presence of high levels of cyanide-insensitive mitochondrial alternative oxidase activity, (iii) cytochrome deficiencies, and (iv) structural abnormalities in the mtDNA. Hence, cytoplasmically transmissible hypovirulence phenotypes found in virus-free strains of C. parasitica from recovering trees may be caused by mutant forms of mtDNA.

DNA primers corresponding to conserved motifs in bacterial repetitive (REP, ERIC, and BOX) elements and PCR were used to show that REP-, ERIC-, and BOX-like DNA sequences are widely distributed in phytopathogenic Xanthomonas and Pseudomonas strains. REP-, ERIC, and BOX-PCR (collectively known as rep-PCR) were used to generate genomic fingerprints of a variety of Xanthomonas and Pseudomonas isolates and to identify pathovars and strains that were previously not distinguishable by other classification methods. Analogous rep-PCR-derived genomic fingerprints were generated from purified genomic DNA, colonies on agar plates, liquid cultures, and directly from lesions on infected plants. REP, ERIC, and BOX-PCR-generated fingerprints of specific Xanthomonas and Pseudomonas strains were found to yield similar conclusions wtih regard to the identity of and relationship between these strains. This suggests that the distribution of REP-, ERIC, and BOX-like sequences in these strains is a reflection of their genomic structure. Thus, the rep-PCR technique appears to be a rapid, simple, and reproducible method to identify and classify Xanthomonas and Pseudomonas strains, and it may be a useful diagnostic tool for these important plant pathogens.

As part of our research to determine phylogenetic relationships of organisms within the phytobacterial species Xanthomonas campestris, we have examined the use of the random amplified polymorphic DNA (RAPD) technique. The objective of this aspect of our research was to determine if a valid cladistic character analysis could be carried out by direct comparison of RAPD products separated on ethidium bromide-stained agarose gels. RAPD products were amplified from 47 Xanthomonas campestris DNA templates using a single oligonucleotide primer. These RAPD products were compared and variation was characterized by Southern analysis of both RAPD products and genomic DNA of the 47 bacterial strains using two cloned RAPD products as probes. Analysis of the data set revealed that the RAPD products were not necessarily homologous or independent, crucial prerequisites for characters to be analyzed in a cladistic phylogenetic analysis. It has been commonly assumed that RAPD variation occurs due to insertion/deletion events or alterations in the primer binding site. Within our data set, we demonstrate absence phenotypes arising from the apparent absence of corresponding loci and also due to the preferred synthesis of alternative RAPD products from unrelated loci. These different types of variation are a reflection of different types of genotypic variation, and direct examination of RAPD products did not allow us to distinguish by which mechanism a particular absence phenotype arose. Although this may not be important for phenetic analyses, for analyses of homologous characters using a cladistic approach it is critical. We also detected unrelated, co-migrating RAPD products and multiple related RAPD products within reaction mixtures. These could both contribute to errors in estimates of similarity, important in any phylogenetic analysis. All of these characteristics of RAPD products should be taken into consideration when RAPD products are used for phylogenetic comparisons.

Double-stranded RNAs (ds RNAs) are thought to be the cytoplasmic determinants responsible for the phenomenon of transmissible hypovirulence in the chestnut blight fungus Endothia parasitica [Murr.] Anderson. The three major ds RNA components associated with the North American hypovirulent strain, Grand Haven 2, were characterized with respect to molecular-hybridization specificity and RNase T1-digestion patterns. The large (L-RNA; approximately 9 kilobase pairs) and middle-sized (M-RNA; approximately 3.5 kilobase pairs) ds RNA components cross-hybridized under stringent conditions and exhibited indistinguishable partial and complete RNase T1 digestion patterns relative to their 5' and 3' termini. These results suggest that M-RNA was derived from L-RNA by an internal deletion event. The small (S-RNA; approximately 1 kilobase pair) RNA was unrelated to L- and M-RNA by these criteria. However, all three ds RNA components contained RNase T1-resistant oligonucleotides at one 5' terminus and at the corresponding 3' terminus of the complementary strand. These RNase T1-resistant species exhibited properties consistent with stretches of poly(uridylic acid) and poly(adenylic acid), respectively. The combined results are discussed in terms of the structural organization of hypovirulence-associated ds RNA molecules and their similarities to "double-stranded" RNA molecules observed in plant and animal cells infected with single-stranded RNA viruses.

Plasmid pRD1 was conjugatively transferred from Escherichia coli to Pseudomonas syringae. Subculturing the transconjugate on a medium that selected for pRD1-determined His+ Kmr resulted in the loss of pRD1 as an extrachromosomal element as detected by agarose gel electrophoresis. DNA hybridization provided evidence for the integration of pRD1 into the P. syringae chromosome.

5 polyhalogenated hydrocarbon natural products isolated from the marine red alga Plocamium spp. were tested for mutagenicity in the Ames reversion assay. All 5 of the compounds induced revertants in Salmonella typhimurium strains TA100 and TA1535, indicating the mutational events involved base substitutions. One of the compounds, designated cross-conjugated ketone, was shown to be almost 200 times more effective as a mutagen than was ethyl methanesulfonate.

The IncP-1 plasmid R68 and variants R68.45 and R68.185 were tested for their chromosome donor ability in a selected recipient of Pseudomonas glycinea PGR12. It was found that variants did not express their selected characteristic of increased donor ability over that of R68 or R68.5, our commonly used donor plasmids. Coinheritance analysis of a variety of crosses provides evidence of a linkage group comprising 11 loci.