A simple and rapid method to determine vegetative compatibility groups in fungi

Vegetative compatibility in fungi reflects phenotypic differences (or similarity) among individuals representing the population of a species (Leslie 1993). Thus, individuals (genotypes) of a fungal species having the same heterokaryon (het) or vegetative incompatibility (vic) loci can fuse to form a heterokaryon (Glass et al 2000). Fungal isolates that form stable heterokaryons are then considered to belong to same vegetative compatible group (VCG). In contrast, isolates that are different at one or some or more of these loci will not anastomose. Rather, programmed cell death or apoptosis occurs in the mycelial cells that are in contact with an isolate representing a different VCG (Anagnostakis 1987, Leslie 1993). In the case of fungi which have coloured or dark mycelium in culture (such as most Botryosphaeriaceae and Cryphonectriaceae) failure to anastomose is observed as a thick barrage line between the two different isolates. For such species, tests in Petri dishes make it relatively easy to determine the VCG’s for a population of isolates and this provides a robust view of population diversity (Burgess et al 2001, van Heerden and Wingfield 2001). For fungi with light coloured mycelium, for example species of Fusarium, barrage zones between isolates having different VCG’s are difficult to discern. In such cases, it is necessary to produce nit mutants to define the individuals in culture (Klittich and Leslie 1988, Swift 2002)

Diplodia scrobiculata found in the southern hemisphere

Diplodia scrobiculata, a latent pathogen of Pinus spp. and other conifers with a limited distribution in the United States, Mexico and southern Europe, has not been reported previously in the southern hemisphere. This is unlike its close relative Diplodia pinea that is found in most parts of the world where pines are native or have been introduced. During an intensive D. pinea survey conducted in Pinus patula plantations in eastern parts of South Africa, a small number of isolates atypical of D. pinea were found. Morphological studies and DNA sequence comparisons showed that these isolates represent D. scrobiculata. Microsatellite analyses suggest that the South African isolates of D. scrobiculata might have originated from California. Pathogenicity tests showed that some of the D. scrobiculata isolates were as pathogenic as those of D. pinea on Pinus radiata and Pinus elliottii

Distribution of Diplodia pinea and its genotypic diversity within asymptomatic Pinus patula trees

Diplodia pinea (= Sphaeropsis sapinea) is an endophytic fungus and opportunistic canker pathogen of Pinus spp. The diversity of this fungus has been studied at broad geographic scales, but little is known regarding its population structure at smaller spatial scales such as within a single tree. This is despite the importance that diversity in a single tree might hold for understanding the biology of the fungus, especially the role of the endophytic or asymptomatic phase in disease development. Moreover there was not information regarding the distribution of the fungus within healthy trees and its persistence. The genotypic diversity of these isolates was investigated using microsatellite markers. Five polymorphic markers were developed and these were used together with eight previously developed markers and vegetative compatibility tests to study the genotypic diversity of D. pinea isolates. In this study, D. pinea was isolated for the first time in the well structured stems of healthy P. patula trees along with branches and cones. From a total of 44 isolates collected from five trees, 39 microsatellite haplotypes and 32 vegetative compatibility groups (VCG's) were identified. The results indicate high genotypic diversity of D. pinea within individual asymptomatic trees which will lead to disease outbreak when trees are physiologically stressed

Diverse sources of infection and cryptic recombination revealed in South African Diplodia pinea populations

This study considers the population diversity and structure of Diplodia pinea in South Africa at different spatial scales from single trees to plantations, as well as comparing infections on healthy and diseased trees. A total of 236 isolates were characterized using 13 microsatellite markers. Analysis of these markers confirmed previous results that D. pinea has a high level of gene and genotypic diversity in South Africa, with the latter values ranging from 6. % to 68. % for the different plantations. The data also reflect a fungus with randomly associated alleles in populations at local plantation scales and for the population as a whole. These results suggest that recombination is occurring in D. pinea and that it most likely has a cryptic sexual state. The study also reveals the sources of endophytic infection and stress related disease out-breaks as diverse infections that have occurred over a long time period. In contrast, wound-associated die-back appears to be caused by clones of the pathogen occurring in narrow time frames

Sources of Diplodia pinea endophytic infections in Pinus patula and P. radiata seedlings in South Africa

Diplodia pinea, an opportunistic and latent pathogen, can significantly affect Pinus productivity worldwide. Despite being studied in South Africa for almost 100years, the source of D. pinea inoculum responsible for seedling infection is unknown. The aim of this study was to determine the role of seed in vertical transmission of D. pinea and to investigate sources of inoculum leading to horizontal transmission to pine seedlings. Surface-disinfected seeds were inoculated with spore and mycelium suspensions of D. pinea to determine its effect on germination. In addition, isolation of the fungus was performed from surface-disinfected seeds, asymptomatic seedlings collected from nurseries, plantations where pines naturally regenerate and recently established fields, to assess transmission and incidence of endophytic D. pinea infections. Inoculation of seeds with D. pinea spore suspensions affected speed and rate of germination. The fungus was isolated from surface-disinfected seeds in only a few instances (2-3%) and was not found in healthy seedlings collected from greenhouses and nurseries, suggesting that vertical transmission of the fungus does not occur or is rare. In contrast, D. pinea was isolated from 40% of seedlings obtained from the understory of mature P. patula trees showing that horizontal transmission from mature to young trees sustains the D. pinea inoculum in South African pine plantations

The infection and diversity of Diplodia pinea in asymptomatic Pinus patula trees

Diplodia pinea (= Sphaeropsis sapinea) is a common latent pathogen in Pine trees in many parts of the world, including South Africa. The aim of this study was to determine the distribution and frequency of endophytic D. pinea infections in Pinus patula trees, seedlings and seeds. The diversity of the endophytic isolates from trees was then determined using 13 microsatellite markers. Isolations of the fungus were successfully made from the main stem, branches and cones of asymptomatic P. patula trees. The genotypic diversity of these isolates was high, ranging between 43% to 68%. The haplotypes in each tree were not spatially grouped, but randomly distributed throughout the tree, indicating numerous individual infections over time. Seedlings grown in areas where mature pines were absent (either in nurseries or in the open field) contained low levels or no D. pinea infection, while those grown in close proximity to mature trees had a 40% incidence of infection. Furthermore, the fungus could only be isolated from 2–3% of surface disinfected seeds. These data indicate that seeds play a limited role in dissemination of D. pinea, although the large amount of seed moved internationally increases the changes of dispersal. The fungus infects plants repeatedly from a young age from inoculum produced on surrounding trees. Latent infections can then persist in the trees for long periods as local infections throughout the tree, even deep in heart of the main stem

Conservation agriculture: a further step in sustainable agricultural intensification in the northern Ethiopian Highlands

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High levels of genetic diversity and cryptic recombination is widespread in introduced Diplodia pinea populations

Introduced populations of organisms typically have reduced diversity compared to those that are native. It is, therefore, unusual that introduced populations of the fungal tree pathogen Diplodia pinea have been shown to have high levels of genetic diversity, even surpassing diversity in some native regions. This is thought to be due to multiple introductions over time or the existence of a cryptic and yet undiscovered sexual cycle. In this study, we consider whether populations of D. pinea in Southern Hemisphere countries have similar patterns of diversity, share some level of genetic identity and how they might be influenced by sexual recombination. A total of 173 isolates from Argentina, Australia, Ethiopia and South Africa were characterized using 12 microsatellite markers. The results show that all these populations have high gene and genotype diversities, with the Australian population having the lowest diversity. Very few private alleles were found, suggesting that isolates from different countries might share a source of introduction. However, based on allele distribution and frequency, each of the populations appeared to be evolving independently. The results showed that in all but the Australian population, alleles are randomly associated, suggesting that widespread sexual recombination has influenced population structure