Louela A. Castrillo

Three genera in the Ceratocystidaceae are the respective symbionts of three independent lineages of ambrosia beetles with large, complex mycangia

The genus Ambrosiella accommodates species of Ceratocystidaceae (Microascales) that are obligate, mutualistic symbionts of ambrosia beetles, but the genus appears to be polyphyletic and more diverse than previously recognized. In addition to Ambrosiella xylebori, Ambrosiella hartigii, Ambrosiella beaveri, and Ambrosiella roeperi, three new species of Ambrosiella are described from the ambrosia beetle tribe Xyleborini: Ambrosiella nakashimae sp. nov. from Xylosandrus amputatus, Ambrosiella batrae sp. nov. from Anisandrus sayi, and Ambrosiella grosmanniae sp. nov. from Xylosandrus germanus. The genus Meredithiella gen. nov. is created for symbionts of the tribe Corthylini, based on Meredithiella norrisii sp. nov. from Corthylus punctatissimus. The genus Phialophoropsis is resurrected to accommodate associates of the Xyloterini, including Phialophoropsis trypodendri from Trypodendron scabricollis and Phialophoropsis ferruginea comb. nov. from Trypodendron lineatum. Each of the ten named species was distinguished by ITS rDNA barcoding and morphology, and the ITS rDNA sequences of four other putative species were obtained with Ceratocystidaceae-specific primers and template DNA extracted from beetles or galleries. These results support the hypothesis that each ambrosia beetle species with large, complex mycangia carries its own fungal symbiont. Conidiophore morphology and phylogenetic analyses using 18S (SSU) rDNA and TEF1α DNA sequences suggest that these three fungal genera within the Ceratocystidaceae independently adapted to symbiosis with the three respective beetle tribes. In turn, the beetle genera with large, complex mycangia appear to have evolved from other genera in their respective tribes that have smaller, less selective mycangia and are associated with Raffaelea spp. (Ophiostomatales).

Identification of Ice-Nucleating Active Pseudomonas fluorescens Strains for Biological Control of Overwintering Colorado Potato Beetles (Coleoptera: Chrysomelidae)

Abstract Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to elevate the supercooling point, the temperature at which freezing is initiated in body fluids, of Colorado potato beetles, Leptinotarsa decemlineata (Say), and to persist in their gut. Adult beetles fed ice-nucleating active strains of Pseudomonas fluorescens, P. putida, or P. syringae at 106 or 103 bacterial cells per beetle had significantly elevated supercooling points, from –4.5 to –5.7°C and from –5.2 to –6.6°C, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was –9.2°C. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88–335 still had significantly elevated supercooling points, indicating that these strains of bacteria were retained. Furthermore, beetle supercooling points were comparable to those observed immediately after ingestion, suggesting that beetle gut conditions were favorable not only for colonization but also for expression of ice-nucleating activity by these two strains. The results obtained from exposure to a single, low dose of either bacterial strain also show that a minimum amount of inoculum is sufficient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also confirmed using a polymerase chain reaction technique that detected ice-nucleating active bacteria by virtue of their ina genes. Application of these ice-nucleating active bacteria to elevate the supercooling point of this freeze-intolerant insect pest could significantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.

Biology of two members of the Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae), recently invasive in the U.S.A., reared on an ambrosia beetle artificial diet

Recent molecular studies have found that the ambrosia beetle Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is a complex of cryptic species, each carrying a different species of symbiotic fungus, in the genus Fusarium, which they farm within galleries inside woody hosts. Several of these beetle species have become invasive pests around the world for attacking and infecting healthy trees with their phytopathogenic fungal symbionts. Diet and rearing protocols were developed for two members of the E. fornicatus species complex, polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB), using sawdust from host trees, allowing collection of data on beetle biology, phenology and sex ratios. Adults developed within 22 days at 24 °C. Single PSHB or TSHB foundresses averaged 32.4 and 24.7 adult female offspring, respectively, and up to 57 and 68 female adults within 6–7 weeks. A strong predictor of the number of offspring in a colony was the number of entry holes. Average sex ratios (% male) for PSHB and TSHB, respectively, were 7.4% and 7.2%. Being haplodiploid, virgin PSHB foundresses were able to produce and mate with male offspring, then subsequently produce female offspring, confirming that they have arrhenotokous reproduction. A cold tolerance study found significant mortality rates among PSHB colonies exposed to −5° or −1 °C but not colonies exposed to 0°, 1° or 5 °C. Given Hamiltons local mate competition (LMC) theory, a number of LMC predictions were violated. PSHB sex ratios were not affected by the number of foundresses; approximately 14% of broods did not contain males; males did not usually eclose before females but eclosed around the same time (22–23 days); and PSHB males were found walking outside of their natal galleries on the trunk of a heavily infested tree in the field. Alternatives to LMC are considered, such as early forms of sociality (maternal care, cooperative brood care), local resource enhancement and kin selection.

Multilocus genotyping of Amylostereum spp. associated with Sirex noctilio and other woodwasps from Europe reveal clonal lineage introduced to the US

Sirex noctilio is a woodwasp of Eurasian origin that was inadvertently introduced to the southern hemisphere in the 1900s and to North America over a decade ago. Its larvae bore in Pinus spp. and can cause significant mortality in pine plantations. S noctilio is associated with a symbiotic white rot fungus, Amylostereum areolatum, which females inject into trees when they oviposit and which is required for survival of developing larvae. We compared the genetic diversity of A. areolatum isolated from S. noctilio and other woodwasps collected from Europe and from northeastern North America to determine the origin of introduction(s) into the United States. Multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed two widespread multilocus genotypes (MLGs) among the European samples, one of which is present in the US. The other two MLGs associated with S. noctilio in the US represented unique haplotypes. These latter two haplotypes were likely from unrepresented source populations, and together with the introduced widespread haplotype reveal multiple A. areolatum MLGs introduced by S. noctilio and indicate possible multiple S. noctilio introductions to North America from Europe. Our results also showed a lack of fidelity between woodwasp hosts and Amylostereum species.

Transstadial Transmission of Pythium in Bradysia impatiens and Lack of Adult Vectoring Capacity

Fungus gnats have been shown to transmit a variety of plant-pathogenic fungi that produce aerial dispersal stages. However, few studies have examined potential interactions between fungus gnats and oomycetes, including Pythium spp. A series of laboratory experiments were conducted to determine whether fungus gnat adults are vectors of several common greenhouse Pythium spp., including Pythium aphanidermatum, P. irregulare, and P. ultimum. An additional objective was to determine whether P. aphanidermatum can be maintained transstadially in the gut of a fungus gnat larva through the pupal stadium to be transmitted by the subsequent adult. Adult fungus gnats did not pick up infectious Pythium propagules from diseased plants and transmit them to healthy plants in any experiment. Species-specific primers and a probe for real-time polymerase chain reaction were developed to detect the presence of P. aphanidermatum DNA in fungus gnat tissue samples. P. aphanidermatum DNA was detectable in the larval and pupal stages; however, none was detected in adult fungus gnats. These results are in agreement with previous studies that have suggested that adult fungus gnats are unlikely vectors of Pythium spp.

First Report of Blight Caused by Sclerotium rolfsii on the Invasive Exotic Weed, Vincetoxicum rossicum (Pale Swallow-Wort), in Western New York

Pale (Vincetoxicum rossicum) and black swallow-wort (V. nigrum) are perennial, twining vines that are increasingly invasive in natural and managed ecosystems in the northeastern United States and southeastern Canada. Both species, introduced from Europe in the 1800s, are listed as noxious weeds or banned invasive species by the USDA-Natural Resource Conservation Service. Observations by C. Southby, a local naturalist, over several years at a meadow populated by pale swallow-wort in Powder Mill Park, Monroe County, NY, revealed a gradual disappearance of pale swallow-wort with restoration of native grasses and some dicotyledonous plant species, in a 6.7-m-diameter area. Diseased swallow-wort plants had extensive yellowing and wilting of foliage, likely due to splitting of the basal stem, with white mycelium throughout the stem and crown; small, reddish brown sclerotia were evident, but roots were not affected. Stem tissue sections from 20 symptomatic plants were vacuum infiltrated with 2% NaOCl for 20 min, then plated onto malt yeast agar and potato dextrose agar amended with 60 mg/liter of penicillin and 80 mg/liter of streptomycin, resulting in development of fast-growing, white mycelium which then formed numerous, irregularly shaped (2 to 4 mm diameter), reddish brown sclerotia at the plate edges. Two individual cultures were identified as S. rolfsii (1) based on size, shape, and color of the sclerotia and presence of characteristic clamp connections in the mycelium. The isolate was suspected to be S. rolfsii var. delphinii due to the reported inability of S. rolfsii to persist in regions with extremely low winter temperatures (4), but molecular data showed otherwise. Sequences of the 18S gene (GenBank JN543690), internal transcribed spacer region (JN543691), and 28S gene (JN543692) of the ribosomal DNA identified the isolate, VrNY, as S. rolfsii (2,3). Pathogenicity tests were conducted with individual 2-month-old seedlings of V. rossicum and V. nigrum grown in steam-sterilized Metromix 360 in SC10 polypropylene conetainers in a growth chamber with a diurnal cycle of 25/20°C, a photoperiod of 14-h light/10-h dark, and fertilized at 3 week intervals. Two independent replications of 12 plants of each species were each inoculated at the stem base with a 4-mm-diameter mycelial agar plug from the growing edge of a colonized plate. The agar plug was held in place with 5 g of sterile sand. Control plants (12 of each species per replication) were treated with sterile agar plugs. Plants for each treatment were placed within a clear plastic bag to maintain 90% relative humidity for 72 h, and then removed from the bags. Disease symptoms developed over 21 days, with >90% of inoculated plants showing symptoms within 2 weeks. Control plants were symptomless. Incidence of mortality was 66 and 60% for V. rossicum and V. nigrum, respectively, by 3 weeks. The fungus reisolated from diseased stem and crown tissue produced characteristic mycelium with irregular sclerotia, consistent with those of S. rolfsii. Since spread of this fungus is based on movement of soilborne sclerotia, this isolate may offer potential as a bio-herbicide for control of swallow-wort in natural ecosystems if the isolate can be demonstrated to have a host range restricted to this invasive weed. References: (1) B. A. Edmunds and M. L. Gleason. Plant Dis. 87:313, 2003. (2) C. E. Harlton et al. Phytopathology 85:1269, 1995. (3) I. Okabe and N. Matsumoto. Mycol. Res. 107:164, 2003. (4) Z. Xu et al. Plant Dis. 92:719, 2008.

Detection of presumptive mycoparasites associated with Entomophaga maimaiga resting spores in forest soils

The fungal pathogen Entomophaga maimaiga can provide high levels of control of the gypsy moth, Lymantria dispar, an important forest defoliator. This fungus persists in the soil as resting spores and occurs naturally throughout many areas where gypsy moth is established. Studies on the spatial dynamics of gypsy moth population have shown high variability in infection levels, and one possible biological factor could be the variable persistence of E. maimaiga resting spores in the soil due to attacks by mycoparasites. We surveyed presumptive mycoparasites associated with parasitized E. maimaiga resting spores using baiting and molecular techniques and identified an ascomycete (Pochonia sp.) and oomycetes (Pythium spp.).

Sleeping Beauties: Horizontal Transmission via Resting Spores of Species in the Entomophthoromycotina

Many of the almost 300 species of arthropod-pathogenic fungi in the Entomophthoromycotina (Zoopagomycota) are known for being quite host-specific and are able to cause epizootics. Most species produce two main types of spores, conidia and resting spores. Here, we present a review of the epizootiology of species of Entomophthoromycotina, focusing on their resting spores, and how this stage leads to horizontal transmission and persistence. Cadavers in which resting spores are produced can often be found in different locations than cadavers of the same host producing conidia. Resting spores generally are dormant directly after production and require specific conditions for germination. Fungal reproduction resulting from infections initiated by Entomophaga maimaiga resting spores can differ from reproduction resulting from conidial infections, although we do not know how commonly this occurs. Reservoirs of resting spores can germinate for variable lengths of time, including up to several months, providing primary infections to initiate secondary cycling based on conidial infections, and not all resting spores germinate every year. Molecular methods have been developed to improve environmental quantification of resting spores, which can exist at high titers after epizootics. Ecological studies of biological communities have demonstrated that this source of these spores providing primary inoculum in the environment can decrease not only because of germination, but also because of the activity of mycopathogens.