Differential patterns of ophiostomatoid fungal communities associated with three sympatric Tomicus species infesting pines in south-western China, with a description of four new species.

Bark beetles and their associated fungi, which cause forest decline and sometimes high mortality in large areas around the world, are of increasing concern in terms of forest health. Three Tomicus spp. (T.brevipilosus, T.minor and T.yunnanensis) infect branches and trunks of Pinusyunnanensis and P.kesiya in Yunnan Province, in south-western China. Tomicus spp. are well known as vectors of ophiostomatoid fungi and their co-occurrence could result in serious ecological and economic impact on local forest ecosystems. Nonetheless, knowledge about their diversity, ecology, including pathogenicity and potential economic importance is still quite rudimentary. Therefore, an extensive survey of ophiostomatoid fungi associated with these Tomicus species infesting P.yunnanensis and P.kesiya was carried out in Yunnan. Seven hundred and seventy-two strains of ophiostomatoid fungi were isolated from the adult beetles and their galleries. The strains were identified based on comparisons of multiple DNA sequences, including the nuclear ribosomal large subunit (LSU) region, the internal transcribed spacer regions 1 and 2, together with the intervening 5.8S gene (ITS) and the partial genes of ß-tubulin (TUB2), elongation factor 1α (TEF1-α) and calmodulin (CAL). Phylogenetic analyses were performed using maximum parsimony (MP) as well as maximum likelihood (ML). Combinations of culture features, morphological characters and temperature-dependent growth rates were also employed for species identification. Eleven species belonging to five genera were identified. These included six known species, Esteyavermicola, Leptographiumyunnanense, Ophiostomabrevipilosi, O.canum, O.minus and O.tingens and four novel taxa, described as Graphilbumanningense, O.aggregatum, Sporothrixpseudoabietina and S.macroconidia. A residual strain was left unidentified as Ophiostoma sp. 1. The overall ophiostomatoid community was by far dominated by three species, representing 87.3% of the total isolates; in decreasing order, these were O.canum, O.brevipilosi and O.minus. Furthermore, the ophiostomatoid community of each beetle, although harbouring a diversity of ophiostomatoid species, was differentially dominated by a single fungal species; Ophiostomacanum was preferentially associated with and dominated the ophiostomatoid community of T.minor, whereas O.brevipilosi and O.minus were exclusively associated with and dominated the ophiostomatoid communities of T.brevipilosus and T.yunnanensis, respectively. Eight additional species, representing the remaining 12.7% of the total isolates, were marginal or sporadic. These results suggested that sympatric Tomicus populations are dominated by distinct species showing some level of specificity or even exclusivity.

Flumorph Is a Novel Fungicide That Disrupts Microfilament Organization in Phytophthora melonis.

ABSTRACT The mechanism of the effects of flumorph (a novel fungicide) was investigated by analyzing alterations of hyphal morphology, cell wall deposition patterns, F-actin organization, and other organelles in Phytophthora melonis. Calcofluor white staining suggested that flumorph did not inhibit the synthesis of cell wall materials, but disturbed the polar deposition of newly synthesized cell wall materials during cystospore germination and hyphal growth. After exposure to flumorph, zoospores were able to switch into cystospores accompanied with the formation of a cell wall, whereas cystospores failed to induce the isotropic-polar switch and did not produce germ tubes but continued the isotropic growth phase. In flumorph-treated hyphae, the most characteristic change was the development of periodic swelling ("beaded" morphology) and the disruption of tip growth. Newly synthesized cell wall materials were deposited uniformly throughout the diffuse expanded region of hyphae, in contrast to their normal polarized patterns of deposition. These alterations were the result of F-actin disruption, identified with the fluorescein isothiocynate (FITC)-phalloidin staining. The disruption of F-actin also was accompanied by disorganized organelles: each swelling of subapical hyphae was associated with a nucleus. Vesicles did not undergo polarized secretion to the apical hyphae, but diffused around nuclei for the subapical growth; thus, the cell wall was thickened with periodic expansion along the hyphae. Upon removing flumorph, normal tip growth and organized F-actin were observed again. These data, as well as data published earlier, suggest that flumorph may be involved in the impairment of cell polar growth through directly or indirectly disrupting the organization of F-actin. The primary site of action by flumorph in the disruption of the F-actin organization is under investigation.