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.

First Report of Aerial Blight of Ruth's Golden Aster (Pityopsis ruthii) Caused by Rhizoctonia solani in the United States.

Ruth's golden aster (Pityopsis ruthii (Small) Small: Asteraceae) is an endangered, herbaceous perennial that occurs only at a few sites along the Hiwassee and Ocoee rivers in Polk County, Tennessee. This species is drought, heat, and submergence tolerant and has ornamental potential as a fall flowering landscape plant. In 2012, we vegetatively propagated various genotypes and established plantings in a landscape at Poplarville, Mississippi. In June and July of 2013, during periods of hot and humid weather, several well-established plants exhibited black or brown necrotic aerial blight symptoms including desiccation of stems and leaves. Blighted leaf samples were surface sterilized (10% commercial bleach, active ingredient 8.25% sodium hypochlorite, 1 min), rinsed in sterile water, air-dried, and plated on 2% water agar amended with 3.45 mg fenpropathrin/liter (Danitol 2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St. Louis, MO). Rhizoctonia sp. was identified based on hyphal morphology and cultures were maintained on potato dextrose agar. Colonies were fast growing, consisting of light tan to brown mycelia and tufts of crystalline aerial hyphae. Within 10 days, brown exudates were present in cultures and there was no pigmented reverse to the agar. Hyphae were a mean of 5.2 µm wide (4.6 to 6.1 µm; n = 10) and each compartment contained three or more nuclei. Hyphae were constricted at septa with right angle branching and no clamp connections, which is typical for Rhizoctonia solani (1). Light- to medium-brown, oblong to irregularly shaped sclerotia measuring 1.2 mm long (0.7 to 2.1 mm) × 0.9 mm wide (0.5 to 1.2 mm; n = 20) were formed in cultures after 3 weeks of growth. Total genomic DNA was extracted from two different colonies grown in potato dextrose broth for 7 days, amplified with PCR using ITS1 and ITS4 primers for amplification of the 18S rDNA subunit (2), the products purified, and sequenced. A consensus sequence of 657 bp was deposited in GenBank (Accession Nos. KF843729 and KF843730) and was 96% identical to two R. solani Kühn ITS sequences in GenBank (HF678125 and HF678122). R. solani was grown on twice autoclaved oats for 2 weeks at 21°C and incorporated into Pro-Mix BX, low fertility soilless medium (Premier Horticulture, Rivière-du-Loup, Quebec, Canada) at 4% (w/w) to inoculate seven P. ruthii plants grown in 10 cm-diameter pots; seven additional plants were grown in the same medium amended with 4% (w/w) sterile oats. Plants were grown in a greenhouse and covered with a plastic dome to maintain high humidity. After 2 weeks, six of the seven inoculated plants exhibited the same aerial blight symptoms as did the original infected plants from the field; none of the control plants developed disease symptoms. Colony morphology and hyphal characteristics as well as the sequence for the ITS region of rDNA from the re-isolated fungus were identical to the original isolate. To our knowledge, this is the first report of R. solani infecting Ruth's golden aster. We are not aware of the disease occurring in wild populations of the plant, but may impact plants grown in the landscape or greenhouse. References: (1) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St Paul, MN, 1991. (2) 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.

First Report of Powdery Mildew on Ruth's Golden Aster (Pityopsis ruthii) Caused by Golovinomyces cichoracearum (Erysiphe cichoracearum).

Ruth's golden aster (Pityopsis ruthii (Small) Small: Asteraceae) is an endangered, herbaceous perennial that occurs only at a few sites along small reaches of the Hiwassee and Ocoee rivers in Polk County, Tennessee. As part of a planned restoration program, Ruth's golden aster has been micropropagated in vitro and acclimatized to greenhouse conditions. In February 2011, several established plants in a greenhouse in Knoxville, TN exhibited signs and symptoms of powdery mildew including growth of white mycelium and conidiophores on the adaxial surface of leaves and slight curling upward of leaf margins. Mycelium was superficial and nipple-shaped appressoria were present. Mycelia, conidiophores, and conidia were removed from several leaves, mounted in water, and examined microscopically. Cylindrical to ovoid conidia (n = 100) lacking fibrosin bodies were borne in chains and had a mean length of 32.0 µm (19.2 to 38.7 µm) and width of 14.9 µm (6.3 to 21.2 µm). The description and dimension of the conidia agreed well with that provided for Golovinomyces cichoracearum (Erysiphe cichoracearum) reported on Coreopsis spp. (1,3) and Cirsium arvense (creeping thistle) (2). The teleomorph was not observed. Total genomic DNA was extracted from infected leaves, amplified with ITS1 and ITS4 primers for the 18S rRNA subunit (4), and visualized on a 2% ethidium bromide agarose gel. An amplicon of fungal origin, approximately 550 bp and smaller than the approximately 700-bp plant ITS amplicon, was excised, purified, and then sequenced. This sequence was deposited in GenBank (Accession No. JF779687) and was 99% identical to two G. cichoracearum accessions (Nos. AB77627 and AB77625). Infected leaves were rubbed on leaves of four healthy plants and healthy leaves were rubbed onto other healthy leaves of two additional plants as controls in the greenhouse. Signs of powdery mildew developed on those plants inoculated with infected leaves after 7 to 10 days and the morphology of the fungus was identical to our previous description. To our knowledge, this is the first report of G. cichoracearum (E. cichoracearum) infecting Ruth's golden aster. We are not aware of the disease occurring in wild populations of the plant, but it does impact the production of micropropagated plants in the greenhouse. References: (1) D. A. Glawe et al. Online publication. doi:10.1094/PHP-2006-0405-01-BR. Plant Health Progress, 2006. (2) G. Newcombe and C. Nischwitz. Plant Dis. 88:312, 2004. (3) T. E. Seijo et al. Online publication. doi: 10.1094/PHP-2006-1214-01-BR. Plant Health Progress, 2006. (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 Inc, New York, 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.

Genetic Analysis of Fungicide-Resistant Sclerotinia homoeocarpa Isolates from Tennessee and Northern Mississippi.

Sclerotinia homoeocarpa is the causal agent of dollar spot disease that reduces the uniformity and aesthetic value of golf putting greens. Fungicide-resistant isolates of S. homoeocarpa were collected from putting greens at 10 locations across Tennessee and northern Mississippi. Genetic diversity among the 60 isolates was investigated using vegetative compatibility, conserved gene sequences, and amplified fragment length polymorphism (AFLP). Six tester strains were paired with Tennessee and northern Mississippi isolates on potato dextrose agar. Some of the 60 isolates were delineated into vegetative compatibility groups, but fungicide resistance could not be associated with a particular vegetative compatibility group. Genetic similarities of isolates at the vegetative compatibility level could be attributed to founder effects. Sequencing the regions of CAD, EF1-α, ß-tubulin, and internal transcribed spacers revealed 100% homology among isolates. Capillary gel electrophoresis and analysis of AFLP fragments indicated 86 to 100% similarity between the isolates. Vegetative compatibility and molecular data indicate that the populations of the pathogen are clonal. Isolates did not cluster according to fungicide resistance during unweighted pair group with arithmetic means analysis, but did appear to cluster according to vegetative compatibility group and location. Although associations could not be made between molecular markers and fungicide resistance, links between vegetative compatibility and AFLP markers may provide a foundation from which other studies could be performed.

Microscopic and Macroscopic Studies of the Development of Puccinia hemerocallidis in Resistant and Susceptible Daylily Cultivars.

Infection and colonization of eight daylily cultivars, which varied in resistance to daylily rust, by Puccinia hemerocallidis was studied macroscopically and microscopically. After germination of urediniospores, appressoria formed at the tip of germ tubes and the fungus penetrated the host through stomatal openings 2 days after inoculation (DAI). Under the infection sites, intercellular hyphae aggregated and formed uredia, which released urediniospores 8 DAI. Resistant cultivars, characterized by the development of rapid death of host cells, were separated into three qualitative categories based on absence and presence of necrotic lesions without or with sporulation. In highly resistant cvs. Prairie Blue Eyes and Bertie Ferris, no macroscopic disease symptoms were observed on leaf surfaces although a few collapsed cells were detected microscopically. Both resistant and moderately resistant reactions were characterized by necrotic lesions with many collapsed cells under infection sites. The difference between these two reactions was that uredia and urediniospores were observed in the moderately resistant cv. Chicago Apache, but not in resistant cvs. Buttered Popcorn and Stella De Oro. Susceptible cultivars, characterized by the absence of a hypersensitive response, were separated into two qualitative categories based on restriction of intercellular hyphal growth that delayed development of uredia and formation of urediniospores. Compared to the susceptible cv. Pardon Me, moderately susceptible cvs. Mary Todd and Chorus Line had a delayed latent period and reduced amount of sporulation. The results indicate that hypersensitive cell death is one of the resistance responses to daylily rust. Necrotic lesions on leaf surfaces are associated with the number of collapsed host cells. Delayed latent period and reduced sporulation that resulted from restriction of intercellular hyphal growth could represent another type of resistance response in the daylily-rust pathosystem.

Comparison of aflatoxigenic and nonaflatoxigenic isolates of Aspergillus flavus using DNA amplification fingerprinting techniques.

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.

Spore Germination, Infection Structure Formation, and Colony Development of Erysiphe pulchra on Dogwood Leaves and Glass Slides.

Spore germination, infection structure formation, and colony development of Erysiphe pulchra on glass slides and leaf disks of a susceptible flowering dogwood line were examined using light and scanning electron microscopes. On both glass slides and leaf disks, germination of conidia started within 2 h after inoculation (hai). One to four germ tubes grew from two poles of a conidium, one or two of the germ tubes formed initial appressoria, and only one of the germ tubes with initial appressoria formed secondary appressoria. However, formation of initial and secondary appressoria was delayed on glass slides (48 and 72 hai, respectively) compared with that on dogwood leaf disks (3 and 24 hai, respectively). Branching hyphae did not grow from germinated conidia on glass slides. However, on dogwood leaf disks, branched hyphae were observed 48 hai. In epidermal cells, the fungus formed compact and globose haustoria. Conidia formation on conidiophores started on leaf disks 7 days after inoculation.

Patterns of evolution in Discula fungi and the origin of dogwood anthracnose in North America, studied using arbitrarily amplified and ribosomal DNA.

The anthracnose epidemic caused by exotic filamentous fungi of the genus Discula threatens the future of the prized flowering (Cornus florida L.) and Pacific (C. nuttalli Aud.) dogwoods in North America. A cross-section of fungi that cause anthracnose in broadleaf temperate trees was characterized using DNA amplification fingerprinting, sequence and secondary structure analysis of the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), and compatibility of hyphal anastomosis. ITS-inferred phylogenies rejected the null hypothesis of only one fungal lineage, by defining four monophyletic and well differentiated groups, corresponding to Discula sp., D. quercina, D. umbrinella and D. destructiva, with the last two species sharing a common and recent ancestor. In turn, they showed that the dogwood pathogen, D. destructiva, did not evolve directly from an indigenous population related to Discula sp. In this study, rDNA spacers that are generally considered important for protein synthesis but are selectively neutral, appeared functionally constrained and subject to selective sequence diversification. Results confirmed the high variability of D. umbrinella and the remarkable homogeneity and exotic nature of D. destructiva at the genetic level, clarified the taxonomy and phylogeny of Discula, and provided clues as to the origin and diversification of dogwood anthracnose-causing fungi.

Variability of Three Isolates of Botrytis cinerea Affects the Inhibitory Effects of Calcium on this Fungus.

ABSTRACT Botrytis cinerea is an economically important pathogen. Epidemiological studies are difficult because of the genetic variability within this species. The objectives of this work were to study the variability and to compare the inhibitory effects of Ca on three isolates of B. cinerea from decayed apple (B) and grape (C and C77:4). Among these isolates, B had the least radial growth but had a sporulation rate 40% higher than that of both C77:4 and C. In situ, isolate C incited the largest decay area in the fruit of two of four apple cultivars examined and had the highest polygalacturonase activity in vitro. Maximum mycelial growth was reached with CaCl(2) at 1 g liter(-1) for isolates B and C77:4 and at 4 g liter(-1) for isolate C. Calcium (CaCl(2)) inhibited polygalacturonase activity at 1 g liter(-1) for C and C77:4 and at 16 g liter(-1) for B. Calcium infiltration reduced decay caused by all three isolates by three to five times. Mycelial DNA analysis showed that 42% of the character loci scored were polymorphic and the greatest similarities were found between B and C77:4. These results support the evidence that the biological and statistical variability in research can be affected by the B. cinerea isolate selected. Despite this variation, Ca treatment of apples reduced decay caused by all three Botrytis cinerea isolates.

Micropropagation of flowering dogwood (Cornus florida) from seedlings.

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 µM stimulated the generation of significantly more useable shoots (≥1 cm) compared to all other concentrations (0.5-22.5 µM tested. Thidiazuron (TDZ) at 0.6 and 1.1 µM supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 µM BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246-1230 µM or continuous lower concentrations (0.5-49.0 µM of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 µM IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.

Sequence signatures from DNA amplification fingerprints reveal fine population structure of the dogwood pathogen Discula destructiva.

Isolates of Discula destructiva Redlin and an undescribed species of Discula, the filamentous fungi that cause anthracnose of flowering (Cornus florida L.) and Pacific (Cornus nuttalli Aud.) dogwoods, were analyzed for genetic variation by nucleic acid scanning with arbitrary mini-hairpin oligonucleotide primers. While the fungal population was highly homogeneous throughout the disease range in eastern and western North America, the generation of arbitrary signatures from amplification profiles (ASAP) distinguished isolates from the northeast, middle and southern Appalachian mountain range, and western United States and Canada. ASAP involves a dual-step arbitrary primer-based amplification procedure that generates a large number of informative allelic characters by amplification of monomorphic DNA fingerprints. ASAP analyses showed a fine fungal population structure consistent with the recent and separate introduction of the pathogen on the west and east coasts of North America.

Molecular phylogeny and DNA amplification fingerprinting of Petunia taxa.

The relationship of five species of Petunia and ten cultivars of the cultivated petunia, Petunia x hybrida, were investigated using DNA-amplification fingerprinting (DAF). Reproducible banding profiles were obtained from P. parodii and P. axillaris DNA from different seed sources. In contrast, other petunias such as P. inflata, P. violacea and P. integrifolia produced variable fingerprints when different plants were examined. However, representative profiles of the variable Petunia taxa were obtained by bulking the leaf tissue from ten different individual plants. Each of ten octamer primers revealed polymorphic loci between taxa. Among a total of 201 bands produced, 146 (73%) loci were polymorphic and distinguished all species and cultivars. Phenetic and cluster analysis using DAF markers separated P. axillaris from P. parodii and distinguished between the violet-flowered species, P. inflata, P. violacea, and P. integrifolia. P. parodii grouped together with the monophyletic set of the ten cultivars of P. x hybrida examined, indicating that it had made a major contribution to the development of these cultivars. Cultivars were distributed within the dendograms by flower color. The results demonstrated the utility of DAF in establishing relationships among closely related species and cultivars of Petunia.

Changes in ovule protein profiles associated with somatic embryogenesis in Cercis canadensis (redbud).

The somatic embryogenic potential of Cercis canadensis (redbud) ovules was compared to changes in ovule protein profiles over time. Ovules collected 82-159 dpa were cultured on a modified SH medium and evaluated after six weeks for the development of somatic embryos. Proteins were extracted from additional ovules and analyzed by SDS-PAGE. Ovules produced somatic embryos from 96-139 dpa and the maximum embryogenic response occurred at 107 dpa. Changes in the staining intensity of six protein bands were associated with changes in embryogenic potential. The intensity 32 and 36 kDa proteins decreased when ovules became competent to produce somatic embryos. The four remaining bands (18, 19, 56, and 94 kDA) increased in intensity from the middle to the end of the sampling period and these changes were associated with the loss of the somatic embryogenic potential.

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis.

Immature embryos from 5 Cladrastis lutea (Michx.) K. Koch (yellowwood) trees were initially cultured on modified Schenk and Hildebrandt medium (SH) containing either 4.5, 9.0, 13.5 or 23 µM 2,4-D. One-third of the explants were transferred to SH medium supplemented with 25.0 µM NAA after 2 and 3 weeks respectively. The remaining explants were incubated on the initial 2,4-D containing media for 6 weeks. Groups of somatic embryos formed directly only at the proximal end of cotyledons; only a few formed as single embryos. The greatest numbers were formed from zygotic embryos explanted from 6-8 weeks post-anthesis and initially cultured on medium containing 9 or 13 µM 2,4-D. However, all treatments supported somatic embryogenesis. In the second year, explants were initially cultured on SH medium containing either 9.0, 13.5, or 23 µM 2,4-D and then transferred to SH medium containing 4.0 µM ABA after 2 or 3 weeks. ABA did not affect the development of somatic embryos. Six of 21 somatic embryos germinated on half-strength SH medium without growth regulators. Three entire plantlets were formed, but only one was established in soil.