Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems in an asexual fungal pathogen

Background Asexual fungi include important pathogens of plants and other organisms, and their effective management requires understanding of their evolutionary dynamics. Genetic recombination is critical for adaptability and could be achieved via heterokaryosis — the co-existence of genetically different nuclei in a cell resulting from fusion of non-self spores or hyphae — and the parasexual cycle in the absence of sexual reproduction. Fusion between different strains and establishment of viable heterokaryons are believed to be rare due to non-self recognition systems. Here, we investigate the extent and mechanisms of cell fusion and heterokaryosis in the important asexual plant pathogen Verticillium dahliae. Results We used live-cell imaging and genetic complementation assays of tagged V. dahliae strains to analyze the extent of non-self vegetative fusion, heterokaryotic cell fate, and nuclear behavior. An efficient CRISPR/Cas9-mediated system was developed to investigate the involvement of autophagy in heterokaryosis. Under starvation, non-self fusion of germinating spores occurs frequently regardless of the previously assessed vegetative compatibility of the partners. Supposedly “incompatible” fusions often establish viable heterokaryotic cells and mosaic mycelia, where nuclei can engage in fusion or transfer of genetic material. The molecular machinery of autophagy has a protective function against the destruction of “incompatible” heterokaryons. Conclusions We demonstrate an imperfect function of somatic incompatibility systems in V. dahliae. These systems frequently tolerate the establishment of heterokaryons and potentially the initiation of the parasexual cycle even between strains that were previously regarded as “incompatible.”

Cytoplasmic Mixing, Not Nuclear Coexistence, Can Explain Somatic Incompatibility in Basidiomycetes

Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.

Ganoderma diversity from smallholder oil palm plantations in peatlands of Kampar District, Indonesia based on mycelia morphology and somatic incompatibility

Hamzah A, Saputra R, Puspita F, Nasrul B, Irfandri, Depari NS. 2021. Ganoderma diversity from smallholder oil palm plantations in peatlands of Kampar District, Indonesia, based on mycelia morphology and somatic incompatibility. Biodiversitas 22: 16-22. Basal Stem Rot disease is caused by the pathogenic fungus Ganoderma boninense which has caused major economic losses in the palm oil industry. Ganoderma boninense has been reported not only infecting crops in the field, but also attacking at the immature phase of the plant even in the nursery. Studies related to Ganoderma diversity in oil palm plantations in Riau, Indonesia have not been widely reported. Ganoderma genetic diversity is important because its provide information regarding the mechanism of infection and the spatial distribution. The Ganoderma spp. isolates were collected from three blocks of smallholder oil palm plantation in Deli Makmur Village, Kampar District, Riau Province, Indonesia and brought to Plant Disease Laboratory, Faculty of Agriculture, Riau University, Pekanbaru, Indonesia. This experimental research was conducted using six Ganoderma isolates (Gan1, Gan2, Gan3, Gan4, Gan5, and Gan6) on the parameters of colony diameter and growth speed, while for the parameters of the morphological diversity of Ganoderma mycelium and the diversity based on somatic incompatibility assay was carried out descriptively. The results of this study show that oil palm plants in smallholder plantations in Kampar District are infected by two groups of Ganoderma based on morphology characteristics using UPGMA dendrogram, but different among isolates based on genetically using somatic incompatibility assay. For the next confirmation, it is necessary to further identify whether the six isolates are different species using molecular identification.

First Report of the Armillaria Root Disease Pathogen, Armillaria gallica, on Douglas-fir (Pseudotsuga menziesii) in Arizona

In August 2010, a mycelial fan (isolate AZ32F) of Armillaria sp. was collected from the root collar of a living Douglas-fir tree on the Mogollon Rim within the Coconino National Forest (approximate location 34°25′31.26″N, 111°20′41.04″W, elevation 2,293 m) in central Arizona. Mycelial fans under the bark of living trees are a sign of pathogenicity, and symptoms of the diseased tree included resinosis, sloughing bark, and thinning crown. The infected tree was located on a south-facing slope with approximately 30% tree cover, dominated by ponderosa pine (Pinus ponderosa), with lesser components of Douglas-fir and Gambel oak (Quercus gambelii). Based on three replications of somatic incompatibility tests against 24 tester isolates representing seven North American Armillaria spp., isolate AZ32F showed 100% intraspecific compatibility (colorless antagonism) with all four A. gallica isolates, 22% compatibility with A. calvescens, and 0% compatibility with the remaining Armillaria spp. Based on GenBank BLASTn of isolate AZ32F sequences, the partial LSU-IGS1 (GenBank Accession No. KF186682) showed 99 to 100% similarity to A. gallica and two other related Armillaria spp. with 99 to 100% coverage, and translation elongation factor-1 alpha (tef-1α) sequences (KC525954) showed 96% similarity to A. gallica (JF895844) with 100% coverage. Thus, isolate AZ32F was identified as A. gallica, based on somatic incompatibility tests and DNA sequences (partial LSU-IGS1 and tef-1α). Although the isolate is identified as A. gallica with similarities to other North American isolates, evidence is mounting that currently recognized A. gallica likely represents a species complex that comprises multiple phylogenetic species (4). Previous surveys in Arizona have noted A. mellea and A. solidipes (as A. ostoyae) (3), but A. gallica has never been previously confirmed in this state. Within North America, A. gallica is commonly reported east of the Rocky Mountains and in West Coast states of the United States, where it infects hardwoods and conifers including Douglas-fir (1,2). Its ecological behavior ranges from saprophyte to weak/aggressive pathogen (1,2). Because damage by A. gallica appears to increase on hosts predisposed by stress (1), further surveys are needed to document its distribution, frequency, and ecological behavior in the southwestern United States, where climate change will likely cause tree stress due to maladaptation. Continued surveys for Armillaria spp. will better determine their potential threat within the geologically and ecologically unique Mogollon Rim of Arizona. References: (1) K. Baumgartner and D. M. Rizzo. Plant Dis. 85:947, 2001. (2) N. J. Brazee and R. L. Wick. For. Ecol. Manage. 258:1605, 2009. (3) R. L. Gilbertson and D. M. Bigelow. J. Arizona-Nevada Acad. Sci. 31:13, 1998. (4) M.-S. Kim et al. Phytopathology 102:S4.63, 2012.

Distribution of Genets of Perenniporia subacida in Stands of Chamaecyparis obtusa (Japanese Cypress) Determined by AFLP Fingerprints and Somatic Incompatibility

The clonal structure of Perenniporia subacida, a wood-rotting basidiomycete, was studied in a 30-year-old stand (site A; 1 ha) and a 43-year-old stand (site B; 0.5 ha) of Chamaecyparis obtusa in northern Kagawa Prefecture, Japan. A total of 110 isolates from decayed trees, stumps, wooden fences, logs, and mycelial mats were analyzed by amplified fragment length polymorphism (AFLP) and somatic incompatibility test (SIT). The results of AFLP, which agreed with those of SIT, revealed that the P. subacida population consisted of at least 17 genets at site A and 6 genets at site B. Individual genets were found in 1 to 9 trees at site A and in 1 to 7 trees at site B. One particular genet had spread over a distance of 70 m. Root contacts were observed among roots of decaying stumps and living trees at both sites. White mycelial mats were often found where roots made contact between stumps and neighboring trees. These results suggest that P. subacida spreads vegetatively over significant distances through root contacts.