Characterization of Diaporthe spp. Associated With Spruce Decline on Colorado Blue Spruce in Michigan.

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.

Inhibition of virulent and hypovirulent Cryphonectria parasitica growth in dual culture by fungi commonly isolated from chestnut blight cankers.

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.

Evaluation of Two Decades of Cryphonectria parasitica Hypovirus Introduction in an American Chestnut Stand in Wisconsin.

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.

Quantification and identification of microorganisms found on shell and kernel of fresh edible chestnuts in Michigan.

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.

Hyperparasites influence population structure of the chestnut blight pathogen, Cryphonectria parasitica.

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.

Antimicrobial Activity of Chestnut Extracts for Potential Use in Managing Soilborne Plant Pathogens.

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 (EC50). Pseudomonas fluorescens was the most sensitive (EC50 = 4.4 µg/µl), Phytophthora cambivora was one of the least inhibited (EC50 = 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.

A 971-bp insertion in the rns gene is associated with mitochondrial hypovirulence in a strain of Cryphonectria parasitica isolated from nature.

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.

Mitochondrial plasmid-like elements in some hypovirulent strains of Cryphonectria parasitica.

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.

The Cryphonectria parasitica mitochondrial rns gene: plasmid-like elements, introns and homing endonucleases.

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.