Defective zoospore encystment and suppressed cyst germination of Phytophthora palmivora caused by transient leaching treatments.

The behaviour of encysting zoospores of Phytophthora palmivora during leaching conditions was studied. Zoospores encysted and germinated successfully on polycarbonate membranes after mechanical agitation. Transient (10 min) leaching treatments with nutrient-free buffer underneath the membranes resulted in abnormal encystment and poor germination. The disruption was greatest when leaching was applied during the first minutes after start of encystment and not observed after 20 min. The early sensitivity of cells to leaching coincided with the period when alkali-resistant cell walls were formed (2-6 min after mechanical agitation). Effects of calcium and organic nutrients on encystment during leaching and germination after these treatments were studied. The disruption of encystment by early leaching treatments, but not the suppression of cyst germination, was overcome by adding calcium chloride during mechanical agitation of zoospores. Leaching with calcium containing buffer resulted in suppressed cyst germination as was the case with buffer alone. Leaching with 0.1% peptone containing buffer promoted consistently high encystment and germination.

Calcium Interference with Zoospore Biology and Infectivity of Phytophthora parasitica in Nutrient Irrigation Solutions.

ABSTRACT Calcium, applied as either CaCl2 or Ca(NO3)2 to water or calcium-free soluble fertilizer solution (Peters 20-10-20 Peat Lite Special), affected several important stages of Phytophthora parasitica zoospore behavior relevant to infection and disease spread. Release of zoospores from sporangia was suppressed by Ca(2+) concentrations in the range of 10 to 50 meq. These concentrations also curtailed zoospore motility; 20 meq of Ca(2+) in fertilizer solution caused all zoospores to encyst within 4 h, whereas 94% of zoospores remained motile in unamended solution. In addition, Ca(2+) in the range of 10 to 30 meq stimulated zoospore cysts to germinate in the absence of an organic nutrient trigger, while suppressing the release of a single zoospore (diplanetism) from cysts that did not germinate. In growth chamber experiments, the amendment of the fertilizer solution with 10 or 20 mM Ca(NO3)2 greatly suppressed infection of flood-irrigated, containerized vinca seedlings in a peat-based mix by motile or encysted zoospores of P. parasitica. These results demonstrate that Ca(2+) amendments interfere with P. parasitica zoospore biology at multiple stages, with compounding effects on epidemiology, and suggest that manipulation of Ca(2+) levels in irrigation water or fertilizer solutions could contribute to management of Phytophthora in recirculating irrigation systems.

Vital fluorochromes as tracers for fungal growth studies.

Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl coumarin (CMAC), and the carbocyamines DiIC18(3) and DiOC18(3). The fungi on which the dyes were tested included Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Idriella bolleyi, Pythium oligandrum, Sclerotium cepivorum and Trichoderma harzianum. Most of the fluorochromes gave good initial staining of mycelia or spores; however, FDA fluorescence faded rapidly during excitation, making it impractical for use. Also, the spores and mycelia of B. cinerea and T. harzianum sometimes gave weak fluorescence with Nile red, and the spores and mycelia of I. bolleyi gave unusually weak fluorescence with Cellufluor. There were other variations of staining among the different dye/fungus combinations, but each fungus showed strong fluorescence at least one dye. Cellufluor, CMFDA, CMAC and, to a lesser extent, CFDA and Nile red, were efficiently translocated into new growth from preloaded spores or mycelia, whereas FDA, DiIC18(3) and DiOC18(3) were not. The extent of translocation ranged from 0.1 to 1.2 mm in germ tubes arising from spores, and from 0.9 to 9.2 mm in mycelia extending from dye-loaded agar blocks. The findings suggest that fluorescent dyes could be used as markers or tracers in studies of fungal growth and differentiation.

Behavioural responses of fungal zoospores.

Fungal zoospores respond to external stimuli in at least four phases of their activity: swimming, encystment, adhesion of the cyst and orientation of the germ tube. Specificity and recognition in these events are described and related to the ecology of zoosporic fungi.

Effects of microwave treatment of soil on growth of birch (Betula pendula) seedlings and infection of them by ectomycorrhizal fungi.

Seedlings of birch (Betula pendula Roth.) were grown for 12 weeks in small volumes of field soils heated in a microwave (MW) oven for different times and then either supplemented or not with inocula of ectomycorrhizal fungi and, in one case, with fresh agricultural soil. Growth of roots and shoots was assessed, and the incidence of different mycorrhizal types, distinguished by colour and morphology, was recorded. Bacterial and fungal populations were determined by dilution plating after MW treatment of soils. In four soils, shoot and root growth were markedly enhanced by MW treatments that raised the soil temperature to 60°C or more, irrespective of the exposure times (20-50 s) needed for this. Shoot dry weights were increased 1·9- to 2·9-fold by MW treatment of two soils from young (11-16 years) birch stands, compared with 8·0- and 11·4-fold for two soils from old birchwoods. But MW treatment of a colliery spoil reduced shoot growth 5-fold. The growth responses were not related to the size of seedlings in untreated soils. The responses coincided with substantial reductions in the total populations of bacteria and fungi. Hebeloma subsaponaceum Karsten developed similar numbers of mycorrhizas from mycelial inoculum added to untreated and MW treated soil. But Lactarius pubescent (Fr. ex Krombh.) Fr. developed significantly more mycorrhizas from inoculum added to MW-treated soil than to untreated soil. This difference between the fungi accords with their classification as, respectively, early stage and late stage in reported mycorrhizal successions on birch. Some mycorrhizal types developed on seedlings from resident inoculum in soil. Their incidence was reduced by different amounts following MW treatment, suggesting differences in heat-tolerance of their propagules. Hebeloma sacchariolens Quel., which forms sclerotium-like bodies and which probably developed from resident inoculum, was unaffected by MW treatment of soil. Some mycorrhizal fungi, notably Thelephora terrestris (Ehrh.) Fr., developed in place of others in soils treated for long times (80-180 s), and they probably developed from airborne spores.

EARLY SENESCENCE OF THE ROOT CORTEX OF AGRICULTURAL GRASSES, AND OF WHEAT FOLLOWING ROOT AMPUTATION OR INFECTION BY THE TAKE-ALL FUNGUS.

Nuclear fluorescence following acridine orange staining was used to assess patterns and rates of death of the root cortex (RCD) in Lolium perenne L., L.×hybridum Hausskn. and Dactylis glomerata L. grown in pathogen-free soil in a glasshouse. The pattern of RCD was as previously described for cereals. The rate of RCD differed significantly between grasses and between cultivates of L. pefenne, but in most instances the cortex was anucleate, except for the innermost cell layer next to the endodermis, in 26 to 27 d old regions of seminal root axes. Root impedance caused by a nylon gauze barrier in soil significantly increased the rate of RCD in one tested cultivar of L. perenne. RCD was more rapid in wheat than in grasses. In 8 d old regions of wheat seminal root axes the cortex contained only 54% of the nuclei initially present. Infection of wheat roots by the take-all fungus, Gaeumannomyces graminis (Sacc.) Arx & Olivier var tritici Walker, caused vascular disruption followed by more rapid RCD than in uninfected roots. Similarly rapid RCD occurred in amputated roots, though in all instances the pattern of RCD was unchanged. The rate of RCD was slower in long than in short lengths of amputated root, perhaps because of remobilization of nutrients from dying cells. Nuclei persisted for at least 10 d in the inner cortex of 2 cm lengths of young roots of wheat, barley and L. perenne buried in soil at 20 °C. The pattern of nuclear deletion from the root pieces was the same as in whole roots, and the rate of RCD was faster in wheat than in barley, as also found for roots attached to plants. All these results demonstrate a consistent pattern of cortical senescence in graminaceous roots and suggest that it is a programmed phenomenon.

DIFFERENTIAL (HOST-SPECIFIC) ACCUMULATION OF ZOOSPORES OF PYTHIUM ON ROOTS OF GRAMINACEOUS AND NON-GRAMINACEOUS PLANTS.

Zoospores of Pythium graminicola Subramanian and P. arrhenomanes Drechs., which characteristically infect the Gramineae, differed from spores of P. aphanidermatum (Edson) Fitzp. and P. ultimum Trow, which have wide host ranges, by accumulating to a greater degree on roots of graminaceous as compared with non-graminaceous plants. These differential responses occurred with wild plants collected from field sites or grown in a glasshouse, but not with cultivated cereals and dicotyledonous plants (Antirrhinum majus L. and Lycopersicon esculentum Mill.) grown in a glasshouse. Differential responses involved mainly encystment and accumulation of encysted spores, rather than taxis. Root mucigel was implicated in zoospore accumulation. Host-differences in this respect did not occur with damaged roots; treatment of roots with methylene blue or periodate (but not alcian blue or Calcofluor) prevented or reduced zoospore encystment without markedly affecting accumulation of motile zoospores.