Mycophagous amoebae in Australian forest soils

Twenty-seven soils, mostly from forested locations in southeastern Australia and Western Australia, were surveyed for the presence of mycophagous vampyrellid amoebae. Characteristic perforations and lysis of conidia of Cochliobolus sativus incubated in 20 of these soils indicated the presence of these amoebae. Isolations of Arachnula impatiens and an amoeba which may be a species of Theratromyxa were obtained from 15 soils. A. impatiens perforated and lysed hyphae and chlamydospores of Phytophthora cinnamomi in culture.     

The relationship of inoculum density of Phytophthora cinnamomi chlamydospores in soil and infection of Eucalyptus marginata seedlings

Chlamydospores of Phytophthora cinnamomi were used as inoculum of known density (ID) in an investigation of the rate of jarrah seed germination and the level of infection of seedlings at the various ID's.Inoculum recovery by soil plating was about 2% of the added density. The germination of the jarrah seedlings was accelerated by the increase of the ID, especially at ID of 250 chlamydospores g^?1 soil. The nominal LD₅₀ value for jarrah seedlings per chlamydospore inoculum was calculated as 13.0 chlamydospores g^?1 soil and the number of viable chlamydospores in the rhizosphere at the highest ID was estimated as 9.1.     

Behaviour of Phytophthora cinnamomi rands in different soils and water regimes

The effect of different soils, nutrient states and water regimes on the growth, sporulation and lysis of mycelial inocula of Phytophthora cinnamomi has been examined. It has been observed that the requirements for chlamydospore and sporangium production in soils are relatively non-specific with respect to soil type, pH, percentage organic matter and the presence or absence of an additional food source. In contrast to chlamydospore production, however, production of sporangia in soil depends on a sufficiently low water suction pressure. In some soils a low percentage water content or a water content well below field capacity did not necessarily inhibit sporangium production. The pathogen was a good competitor for pieces of both fresh and rotting Castanea sativa radicles. Trichoderma viride appeared to play a significant role in soil by lysing hyphae of P. cinnamomi and inducing it to produce oospores.     

Fine structure of a new mycophagous amoeba and its feeding on Cochliobolus sativus

An unidentified mycophagous soil amoeba is described. The pigmented soil-borne fungus Cochliobolus sativus and four other fungal species, both pigmented and hyaline, were utilized as food. Spores were ingested and lysed within digestive vacuoles by general wall erosion. This contrasts with the wall perforation mechanism described for other mycophagous amoebae. Ultrastructural studies of trophozoites showed that large quantities of electron dense granules were released into the digestive vacuoles during fungal cell lysis. These were incorporated into the amoebal protoplast. Bacteria were commonly present in the amoebal protoplasts and within digestive vacuoles. Their possible role as endosymbionts is discussed.     

Soil Vampyrellid amoebae that cause small perforations in conidia of Cochliobolus sativus

The parasitic habits of two mycophagous amoebae, members of the Vampyrellidae isolated from soil, were studied under laboratory conditions. The amoeboid organisms resembled Theratromyxa weberi and Vampyrella vorax. Both organisms lysed conidia of Cochliobolus sativus and chlamydospores o Thielaviopsis hasicola within digestive cysts. Perforations 1 μm dia or less were observed in walls of the lysed fungus spores. The mycophagous Theratromyxa sp. and V. vorax were differentiated in laboratory culture chambers on the basis of morphology, encystment, excystment and diameter of perforations produced in walls of conidia of C. sativus. Both organisms differed from a previously-described species of Vampyrella which causes large perforations and annular depressions in spore walls.     

Effect of organic matter on the survival of Phytophthora cinnamomi rands in soil

Soil organic matter collected from beneath an unburnt stand of Eucalyptus marginata was added in increasing amounts to lateritic soil. Phytophthora cinnamomi incubated in soils containing 50% or more organic matter was extensively lysed, and many of the sporangia produced were abortive.With increasing organic matter there is an increase in nutrient concentrations and in the microbial population and it is suggested that these factors are the basis of the antagonism.     

Evaluation of techniques for quantitative detection of Phytophthora cinnamomi

Three baiting techniques and a sieving technique were evaluated for the quantitative detection zof Phytophthora cinnamomi Rands. Baiting with seedlings of Lupinus angustifolius L. (blue lupins), cotyledons of Eucalyptus sieberi F. Muell. and pear fruits cv. Packham's Triumph, detected one chlamydospore introduced into 50 g of either sand or soil. Sieving detected one chlamydospore in 50 g sand and also in 5 g soil. In naturally-infested soil, diluted with a similar non-infested soil, P. cinnamomi was detected at dilutions of $\text{1}\text{8}$ and $\text{1}\text{128}$ by the sieving and the baiting techniques, respectively. In general, pears were the most satisfactory bait for the detection and isolation of P. cinnamomi. The potential of baiting techniques for estimating numbers of propagules of P. cinnamomi in naturally-infested soil is discussed.     

Infection of wheat seminal roots by varieties of Phialophora radicicola and Gaeumannomyces graminis

The growth of isolates of Phialophora radicicola var. radicicola, P. radicicola var. graminicola, Gaeumannomyces graminis var. graminis, G. graminis var. tritici and Leptosphaeria narmari was compared on the coleoptiles and roots of wheat seedlings. Fungal growth was measured as the extent and density of dark runner hyphae. All except P. radicicola var. graminicola grew on coleoptiles and all grew on roots although only G. graminis var. tritici extensively colonized the root stele. Growth rate on roots was positively correlated with that on agar, P. radicicola var. graminicola and L. narmari growing at about half the rate of the other fungi; hyphal density was high for P. radicicola var. graminicola but relatively low for the other fungi. For P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici growing from buried inocula, the extent and density of hyphae up roots towards the seed was similar to that down, but G. graminis var. tritici caused chocolate-brown stelar discoloration up roots only.Root invasion by P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici was described from sections. Each gave a different pattern of hyphae and host response within an inoculum layer, and progressive changes occurred away from the inoculum. Studies of the rate of penetration by each fungus and the rate and pattern of death of cortical cells explained the differences between fungi. G. graminis var. tritici penetrated living cells in advance of other soil micro-organisms, and hence by hyaline hyphae inducing much lignituber formation as a host resistance reaction. P. radicicola var. graminicola penetrated only senescent or dead cells in association with other soil microorganisms, and hence by dark hyphae, inducing little lignituber formation. P. radicicola var. radicicola was intermediate in all these respects. The high hyphal density of P. radicicola var. graminicola was due to the colonization of cortical cells and spaces by dark, clearly visible, rather than hyaline hyphae, which are invisible in unstained roots. Cell death in the outer cortex explained the observed progressive restriction of growth by all fungi to the inner cortex with increasing distance from the inoculum. Spread by G. graminis var. tritici up roots was ectotrophic relative to the stele but down roots hyphae spread rapidly within the stele. Stelar reactions suggested as resistance mechanisms occurred up roots only. Their absence down roots is attributed to infection disrupting stelar transport.     

Production of chlamydospores and conidia by Trichoderma spp in liquid and solid growth media

Isolates of Trichoderma spp grew and produced chlamydospores as well as conidia in molasses-corn steep liquor (M-CSL), sucrose nitrate (SN), and glucose tartrate (GT) media. In M-SCL, isolates of T. hamatum, T. viride, and T. harzianum formed 10.4, 5.9 and 1.1 × 10⁸ chlamydospores g^?1 dry weight of mycelium. Fewer chlamydospores formed in SN and GT. Although T. harzianum formed the least number of chlamydospores, it produced the highest number of conidia in all three media. Molasses-corn steep liquor was superior to SN or GT in supporting development of both spore types. Spore production was not influenced by initial pH of the media or by continuous maintenance of the media at pH 4 or 7. Equal numbers of chlamydospores were formed in liquid media incubated in shake or static culture. Conidia formation, however, was stimulated in static culture. Chlamydospores and conidia of several naturally occurring isolates and induced biotypes of Trichoderma spp were abundantly produced on a variety of solid substrates moistened with liquid nutrients or water, preferably at pH 4. Bran, cornmeal, and peanut hull meal were better than eight other solid substrates for production of chlamydospores and conidia. A u.v.-induced, benomyl-resistant biotype of T. viride (T-1-R9) formed 22 × 10⁷ and 18 × 10⁸ chlamydospores and conidia, respectively, g^?1 of bran-SN. The ratio of conidia to chlamydospores was always greater in solid than liquid media. In solid media, 10 times more conidia than chlamydospores were formed, whereas in liquid only two or three times more were formed. Chlamydospores from liquid and solid fermentations were viable (ca 80%) and fresh chlamydospores germinated well (ca 75%) on nutrient agar. Although dried chlamydospores were viable, as determined with tetrazolium bromide, their germination on agar was poor (ca 8%).     

Effect of phosphonate on the rhizosphere microflora and the development of root rot (Phytophthora cinnamomi) in avocado (Persea americana) and pepper-corn (Schinus molle) tree seedlings

Summary Application to soil of 1 g (recommended rate) or 10 g l^-1 of phosphonate did not affect the numbers of bacteria and fungi nor the proportions of actinomycetes and fungi antagonistic to Phytophthora cinnamomi. Foliar phosphonate applications to avocado seedlings (Persea americana) did not affect microbial numbers or the proportions of microbes in the rhizosphere capable of antagonizing P. cinnamomi. Mycelium of P. cinnamomi and zoospores of P. palmivora did not appear to respond to diffusates from excised roots of phosphonate-treated avocado and pepper-corn tree (Schinus molle) seedlings, respectively. However, less extensive lesions were observed on the roots of fungicide-treated avocado and pepper-corn tree seedlings exposed to P. cinnamomi and P. palmivora, respectively. The reduction in P. cinnamomi infection on pepper-corn tree seedlings appears to be brought about by additive rather than interactive effects of the resident soil microflora and foliar-applied phosphonate.     

Phytophthora cinnamomi: Effects of herbicides on radial growth,sporangial production,inoculum potential and root disease in Pinus radiata

An epidemic of severe root rot of Pinus radiata seedlings caused by Phytophthora cinnamomi in the 1970's at Benalla Nursery, 220 km north-east of Melbourne, was followed by a rapid disappearance of the disease in 1981. This phenomenon coincided with the application of simazine and propazine for weed control between 1971 and 1980 and glyphosate and chlorthal dimethyl after 1980. In laboratory and greenhouse tests, chlorthal dimethyl and glyphosate significantly (P <0.01) reduced radial growth. sporangial production and inoculum potential of P. cinnamomi, whereas propazine and simazine exhibited mild fungitoxic properties (in vitro), and had a stimulatory effect on the production of sporangia and inoculum potential of the pathogen.     

Ultrastructure of developing chlamydospores of Fusarium solani f. Cucurbitae in vitro

Propagules from macroconidial cultures of Fusarium solani f. cucurbitae in vitro were examined at intervals during chlamydospore formation by transmission electron microscopy. There was a gradual breakdown of the original macroconidial cell wall. Simultaneously, synthesis of new cell wall material occurred. The significance of autolytic cell wall degradation as it affects the morphology of chlamydospores formed from macroconidial cells in soil is discussed.     

Solar-heating soil for control of damping-off diseases

Solar-heating (45–52°C) moist soil under 50μm thick clear plastic sheeting during summer (Jan.–Feb.) significantly reduced pre-emergence damping-off disease of Pinus radiata D. Don (radiata pine) and Eucalyptus obliqua L'Herit (messmate and stringybark) seed, and also post-emergence mortality among P. radiata seedlings.Phytophthora cinnamomi Rands, Fusarium oxysporum Snyder and Hansen, and Pythium sp. could not be re-isolated from artificially inoculated pine roots after exposure to the solar-heating treatment. Natural infectious propagules of P. cinnamomi were also undetectable in solar-heated soil for up to 16 months following treatment, though infectious propagules of Pythium were detected at low level. The treatment also controlled 11 weed species. Solar-heating a potting mixture temporarily suppressed disease incidence in nursery stock, possibly due to an increase in antagonistic microorganisms.Solar-heating moist soil appears to be particularly attractive in forest nursery practice, as it provides a simple, effective, non-toxic and non-polluting technique for control of soil-borne diseases and weed species.     

Growth and interactions of arbuscular mycorrhizal fungi in soils from limestone and acid rock habitats

Arbuscular mycorrhizal (AM) development in different soil types, and the influence of AM fungal hyphae on their original soil were investigated. Plantago lanceolata, which can grow in soils of a very wide pH range, was grown in two closely related limestone soils and an acid soil from rock habitats. Plants were colonised by the indigenous AM fungal community. The use of compartmented systems allowed us to compare soil with and without mycorrhizal hyphae. Root colonisation of P. lanceolata was markedly higher in the limestone soils (30-60%) than in the acid soil (5-20%), both in the original habitat and in the experimental study. Growth of extraradical AM fungal hyphae was detected in the limestone soils, but not in the acid soil, using the signature fatty acid 16:1ω5 as biomass indicator. Analysis of signature fatty acids demonstrated an increased microbial biomass in the presence of AM fungal hyphae as judged for example from an increased amount of NLFA 16:0 with 30 nmol g⁻¹ in one of the limestone soils. Bacterial activity, but not soil phosphatase activity, was increased by around 25% in the presence of mycorrhizal hyphae in the first harvest of limestone soils. AM fungal hyphae can thus stimulate microorganisms. However, no effect of AM hyphae was observed on the soil pH or organic matter content in the limestone soils and the available P was not depleted.     

Suppressiveness of 18 composts against 7 pathosystems: Variability in pathogen response

Compost is often reported as a substrate that is able to suppress soilborne plant pathogens, but suppression varies according to the type of compost and pathosystem. Reports often deal with a single pathogen while in reality crops are attacked by multiple plant pathogens. The goal of the present study was to evaluate the disease suppression ability of a wide range of composts for a range of plant pathogens. This study was conducted by a consortium of researchers from several European countries. Composts originated from different countries and source materials including green and yard waste, straw, bark, biowaste and municipal sewage. Suppressiveness of compost-amended (20% vol./vol.) peat-based potting soil was determined against Verticillium dahliae on eggplant, Rhizoctonia solani on cauliflower, Phytophthora nicotianae on tomato, Phytophthora cinnamomi on lupin and Cylindrocladium spathiphylli on Spathiphyllum sp., and of compost-amended loamy soil (20% vol./vol.) against R. solani on Pinus sylvestris and Fusarium oxysporum f. sp. lini on flax. From the 120 bioassays involving 18 composts and 7 pathosystems, significant disease suppression was found in 54% of the cases while only 3% of the cases showed significant disease enhancement. Pathogens were affected differently by the composts. In general, prediction of disease suppression was better when parameters derived from the compost mixes were used rather than those derived from the pure composts. Regression analyses of disease suppression of the individual pathogens with parameters of compost-amended peat-based mixes revealed the following groupings: (1) competition-sensitive: F. oxysporum and R. solani/cauliflower; (2) rhizosphere-affected: V. dahliae; (3) pH-related: P. nicotianae; and (4) specific/unknown: R. solani/pine, P. cinnamomi and C. spathiphylli. It was concluded that application of compost has in general a positive or no effect on disease suppression, and only rarely a disease stimulating effect.     

Increased abundance of arbuscular mycorrhizal fungi in soil coincides with the reproductive stages of maize

Arbuscular mycorrhizal (AM) fungi are recognized for their positive effects on plant growth, playing an important role in plant P nutrition. We used C16:1cis11 and C18:1cis11 fatty acid methyl ester (FAME) biomarkers to monitor the dynamics of AM fungi during the reproductive stages of maize (Zea mays L.) grown at high yield in Nebraska, USA. Two fields with four different levels of P availability were sampled throughout the reproductive stages. Chambers, made of PVC enclosed mesh fabric to allow passage of roots and hyphae (+R) or hyphae alone (-R) and amended with either KH₂PO₄(+P) or distilled water (-P), were installed in the field at tasselling and removed after three, six and nine weeks. Our objectives were (i) to provide evidence for C allocation to AM fungi during the reproductive stages of high productivity maize and (ii) to link AM fungal growth dynamics with changes in soil P availability. We observed that initial AM FAME concentration was lower at sites with a high availability of P. During the reproductive growth of maize, AM biomarkers increased inside the chambers and were consistent with the biomarker increase observed in adjacent field soil. This confirms that there is C allocation from the plant to the symbiont during the reproductive stages of maize. We also observed a reduction in available P in +R and -R chambers. This observation implies that hyphae were as efficient as roots and hyphae in reducing the P concentration in chambers. These results demonstrate that AM fungi are active during the reproductive growth stages of maize and may benefit high productivity maize crops by facilitating P uptake.     

Trapping of phosphate solubilizing bacteria on hyphae of the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM 197198

A simple method is described for trapping phosphate solubilizing bacteria (PSB) strongly attached to the hyphae of the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis (Ri). Bacteria were isolated from the hyphosphere of mycorrhizal leek plants growing on Turface previously inoculated with soil suspensions, obtained from the mycorrhizosphere of mycorrhizal plants growing in agricultural settings or maple forests in Quebec, Canada. Among the best PSB strongly attached to the hyphae of Ri, 26 isolates belonged to Burkholderia spp. and one was identified as Rhizobium miluonense. Four hyphobacteria exhibiting high potential of inorganic and organic P mobilization were further compared with four equivalent mycorrhizobacteria directly isolated from mycorrhizospheric soils sampled. In general, hyphobacteria were superior in mobilizing P from hydroxyapatite and from a low reactivity igneous phosphate rock from Quebec. Release of gluconic acid or the product of its oxidation 2-ketogluconic acid, are the main mechanisms involved in P solubilization. In a two compartments Petri plate system, Ri extraradical hyphal exudates, supported PSB growth and activity. In the absence of PSB Ri showed a negligible P solubilization activity. In the presence of PSB a substantial increase in P mobilization was observed, and the superiority of hyphobacterial activity was also observed under this system. Our results suggest that in developing a bioinoculant based on selected PSB, their interaction with AMF hyphae should not be overlooked.     

Fluorescence microscopy to study colonization of conidia and hyphae of Cochliobolus Sativus by soil microorganisms

Epi-fluorescence microscopy facilitated observations of the colonization of hyphae and conidia of C. sativus and other plant pathogenic fungi in soil. Of six fluorochromes examined, acridine orange and europium chelate effectively differentiated cells of bacteria and actinomycetes from the melanized fungal structures. Under u.v. radiation, the colonizing organisms fluoresced intensively while the pigmented conidia appeared dark. Periodic observations of hyphal fragments and conidia of C. sativus incubated in nonsterile soil revealed rapid colonization of their surface primarily by bacteria. Whereas up to 80% of the hyphal fragments of C. sativus were lysed after 32 days' incubation in soil, the conidia remained intact. However, disorganization of protoplasts and disappearance of conidial septa were evident after the fourth week. After 2, 3 and 5 weeks incubation of the plant pathogens in soil, no significant difference was noticed between microbial colonization of conidia on 8.0 μm pore size “Nucle-pore” membranes and those placed directly on the soil surface.     

Soil fungi as food for giant amoebae

Feeding trials were carried out to assess the ability of a giant vampyrellid soil amoeba to attack and lyse spores of fungi. Of 24 species of fungi studies, 15 were perforated in the same manner as was reported for Cochliobolus sativus. This giant amoeba is nutritionally versatile and can feed on bacteria, flagellates, blue green algae, diatoms and nematodes. Seven other soil amoebae failed to lyse conidia of C. sativus.     

Hyphal density as a measure of suppression of Gaeumannomyces graminis var. tritici on wheat roots

Runner hyphae of Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici Walker on seminal roots of wheat seedlings were photographed and their length measured. As well, their length was estimated using the line-intercept method. The correlation of 0.904 between measured and estimated lengths of hyphae was highly significant. This line intercept method was used to estimate the density (length/unit area) of hyphae on roots of plants growing in the presence and absence of a soil suppressive to G. graminis var. tritici. Estimations were made eight times during 28 days growth at 15°C. In fumigated soil (non-suppressive) inoculated with 0.1% ground oat grain infested with G. graminis var. tritici, the density of hyphae on roots started to increase at five days compared with 15 days when soil there was a 10.8% cover of the root surface after 15 days when the hyphae had reached maximum density. Suppression to G. graminis var. tritici is normally detected by a difference in disease rating of roots at 28 days but this study has shown that suppression can be demonstrated by the difference in the density of hyphae if roots are examined between seven and 19 days.