Evidence for high gene flow, nonrandom mating, and genetic bottlenecks of Ganoderma boninense infecting oil palm (Elaeis guineensis Jacq.) plantations in Malaysia and Indonesia.

Ganoderma boninense, the causal agent of basal stem rot (BSR) disease, has been recognized as a major economic threat to commercial plantings of oil palm (Elaeis guineensis Jacq.) in Southeast Asia, which supplies 86% of the world's palm oil. High genetic diversity and gene flow among regional populations of 417 G. boninense isolates collected from Sabah, Sarawak, and Peninsular Malaysia (Malaysia) and Sumatra (Indonesia) were demonstrated using 16 microsatellite loci. Three genetic clusters and different admixed populations of G. boninense across regions were detected, and they appeared to follow the spread of the fungus from the oldest (Peninsular Malaysia and Sumatra) to younger generations of oil palm plantings (Sabah and Sarawak). Low spatial genetic differentiation of G. boninense (FST = 0.05) among the sampling regions revealed geographically nonrestricted gene dispersal, but isolation by distance was still evident. Analysis of molecular variance (AMOVA) confirmed the little to no genetic differentiation among the pathogen populations and the three genetic clusters defined by STRUCTURE and minimum spanning network. Despite G. boninense being highly outcrossing and spread by sexual spores, linkage disequilibrium was detected in 7 of the 14 populations. Linkage disequilibrium indicated that the reproduction of the fungus was not entirely by random mating and genetic drift could be an important structuring factor. Furthermore, evidence of population bottleneck was indicated in the oldest oil palm plantations as detected in genetic clusters 2 and 3, which consisted mainly of Peninsular Malaysia and Sumatra isolates. The population bottleneck or founding event could have arisen from either new planting or replanting after the removal of large number of palm hosts. The present study also demonstrated that migration and nonrandom mating of G. boninense could be important for survival and adaptation to new palm hosts.

Genetic diversity and gene flow amongst admixed populations of Ganoderma boninense, causal agent of basal stem rot in African oil palm (Elaeis guineensis Jacq.) in Sarawak (Malaysia), Peninsular Malaysia, and Sumatra (Indonesia).

In 1911 and 1917, the first commercial plantings of African oil palm (Elaeis guineensis Jacq.) were made in Indonesia and Malaysia in Southeast Asia. In less than 15 years, basal stem rot (BSR) was reported in Malaysia. It took nearly another seven decades to identify the main causal agent of BSR as the fungus, Ganoderma boninense. Since then, research efforts have focused on understanding G. boninense disease epidemiology, biology, and etiology, but limited progress was made to characterize pathogen genetic diversity, spatial structure, pathogenicity, and virulence. This study describes pathogen variability, gene flow, population differentiation, and genetic structure of G. boninense in Sarawak (Malaysia), Peninsular Malaysia, and Sumatra (Indonesia) inferred by 16 highly polymorphic cDNA-SSR (simple sequence repeat) markers. Marker-inferred genotypic diversity indicated a high level of pathogen variability among individuals within a population and among different populations. This genetic variability is clearly the result of outcrossing between basidiospores to produce recombinant genotypes. Although our results indicated high gene flow among the populations, there was no significant genetic differentiation among G. boninense populations on a regional scale. It suggested that G. boninense genetic makeup is similar across a wide region. Furthermore, our results revealed the existence of three admixed genetic clusters of G. boninense associated with BSR-diseased oil palms sampled throughout Sarawak, Peninsular Malaysia, and Sumatra. We postulate that the population structure is likely a reflection of the high genetic variability of G. boninense populations. This, in turn, could be explained by highly successful outcrossing between basidiospores of G. boninense from Southeast Asia and introduced genetic sources from various regions of the world, as well as regional adaptation of various pathogen genotypes to different palm hosts. Pathogen variability and population structure could be employed to deduce the epidemiology of G. boninense, as well as the implications of plantation cultural practices on BSR disease control in different regions.

Behavioral Responses of Pityophthorus juglandis (Coleoptera: Curculionidae: Scolytinae) to Volatiles of Black Walnut and Geosmithia morbida (Ascomycota: Hypocreales: Bionectriaceae), the Causal Agent of Thousand Cankers Disease.

Thousand cankers disease (TCD) is a pest complex formed by the association of the walnut twig beetle (WTB), Pityophthorus juglandis Blackman (Coleoptera: Curculionidae: Scolytinae), with the fungal pathogen Geosmithia morbida Kolarík, Freeland, Utley and Tisserat (Ascomycota: Hypocreales: Bionectriaceae). Current monitoring and detection efforts for WTB rely on a pheromone lure that is effective over a limited distance while plant- and fungal-derived volatiles that may facilitate host location remain poorly understood. In this study, we test the hypothesis that adult beetles are attracted to volatiles of black walnut, Juglans nigra L. (Juglandaceae), and the pathogen, G. morbida. We measured the response of beetles to head-space volatiles collected from leaves and stems of 12 genotypes of black walnut and found genotypic variation in the attractiveness of host trees to adult WTB. Volatile profiles of the most attractive genotypes contained more α-pinene and ß-pinene, and adult beetles were attracted to both of these compounds in olfactometer bioassays. In bioassays, we also demonstrated that adult WTB are attracted to volatiles of G. morbida. These findings suggest that, in addition to the aggregation pheromone, dispersing WTB potentially use host plant and fungal volatiles to locate suitable larval hosts. Finally, we conducted a field experiment to determine the extent to which ethanol, a common attractant for bark beetles, and limonene, a known bark beetle repellent, influence the behavior of adult WTB to pheromone-baited traps. Although ethanol did not increase trap capture, WTB were repelled by limonene, suggesting that this compound could be used to manipulate and manage WTB populations.

First Report of Geosmithia morbida in North Carolina: The Pathogen Involved in Thousand Cankers Disease of Black Walnut.

In the past decade, black walnut (Juglans nigra) trees throughout western North America have suffered from widespread branch dieback and canopy loss, causing substantial tree mortality (2,3). The fungus, Geosmithia morbida, vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, has been associated with this devastating disease known as Thousand Cankers Disease (TCD) (2,3). In August of 2012, branch samples from TCD symptomatic black walnut trees (5 to 10 cm in diameter and 15 to 30 cm long) were collected on the North Carolina side of the Great Smoky Mountain National Park (GRSM) in Cataloochee Cove (35°37.023' N, 83°07.351' W) and near the Big Creek Campground (35°45.290' N, 83°06.473' W), in Haywood County. Five symptomatic trees near the Big Creek Campground and three from Cataloochee Cove displayed typical TCD signs including progressive crown thinning, branch flagging, and branch dieback; however, insect holes were not observed. Samples were double bagged in Ziploc plastic bags, sealed in a 19-liter plastic bucket, and transported to the University of Tennessee. Outer bark was removed from the samples and small, elliptical, necrotic cankers were observed. Wood chips (3 to 4 mm2) from cankers were excised and placed on 1/10 strength potato dextrose agar amended with 30 mg/liter streptomycin sulfate and 30 mg/liter chlortetracycline HCL and incubated on a 12-h dark/light cycle at 22°C for 5 to 7 days. Fungal isolates were tentatively identified as G. morbida by using culture morphology, and characteristics of conidiophores and conidia (2). The isolated fungus from the Cataloochee Cove location was grown in 1/10 strength potato dextrose broth at room temperature for 2 weeks. Isolates from Big Creek Campground were contaminated and were not analyzed further. Fungal colonies were tan to light yellow. Conidia were tan, subcylindrical, and catenulate. Conidiophores were multibranched, verticillate, and verrucose. To verify the morphological data, DNA was extracted from fungal mycelia using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's published protocol. Isolates from Cataloochee Cove were characterized using ITS1 and ITS4 universal primers (4). The putative G. morbida isolate (GenBank Accession No. KC461929) had ITS sequences that were 100% identical to the G. morbida type isolate CBS124663 (FN434082.1) (2). Additionally, fungal DNA from Cataloochee Cove was amplified using G. morbida-specific microsatellite loci (GS04, GS27, and GS36) (1). PCR products were analyzed with the QIAxcel Capillary Electrophoresis System (Qiagen) and were similar to those previously published (2). To date, all confirmed cases of TCD in the native range of black walnut have been in urban areas, along rural roadsides and/or fence rows. The report in North Carolina is the first finding of G. morbida, the causal agent of TCD, in a forest setting. References: (1) D. Hadziabdic et al. Conserv. Genet. Resources 4:287, 2012. (2) M. Kolarik et al. Mycologia 103:325, 2011. (3) N. Tisserat et al. Plant Health Progr. doi:10.1094/PHP-2011-0630-01-BR, 2011. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

Analysis of genetic diversity in flowering dogwood natural stands using microsatellites: the effects of dogwood anthracnose.

Flowering dogwood (Cornus florida L.) populations recently have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48 to 98%, raising the concern that genetic diversity has been reduced significantly. Microsatellite data were used to evaluate the level and distribution of genetic variation throughout much of the native range of the tree. Genetic variation in areas affected by anthracnose was as high as or higher than areas without die-offs. We found evidence of four widespread, spatially contiguous genetic clusters. However, there was little relationship between geographic distance and genetic difference. These observations suggest that high dispersal rates and large effective population sizes have so far prevented rapid loss of genetic diversity. The effects of anthracnose on demography and community structure are likely to be far more consequential than short-term genetic effects.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).