Modelling the growth of Trichoderma virens with limited sampling of digital images.

AIMS: Using limited digital image sampling, a model of fungal growth in soil that considers both hyphal production and lysis was constructed for two strains of Trichoderma virens over a range of four temperatures. MATERIALS AND METHODS: A growth model was developed by fitting the radial cross sectional data with a modified form of the Ratkowsky equation to determine maximum growth rate and a modified Arrhenius equation to determine maximal rate of decrease in area covered by mycelia. The parameters obtained from a combined equation were then verified by using the data obtained from the whole colony to determine the appropriateness of the model. CONCLUSIONS: Using a limited data set and a combination of the Ratkowsky and Arrhenius equations, the mycelial coverage of the T. virens colony was determined, relating microscopic hyphal growth to macroscopic colony growth. This model was sufficiently robust to predict growth across four temperatures for a genetically modified and wild-type strain of T. virens. SIGNIFICANCE AND IMPACT OF STUDY: By using simple assumptions for the increase and eventual decline in fungal growth on a resource-limited medium, this model constructs an initial framework onto which additional parameters such as nutrient consumption could be incorporated for prediction of fungal growth.

Visual and Infrared Assessment of Root Colonization of Apple Trees by Phymatotrichopsis omnivora.

Root systems of 5-year-old, trellised apple trees (Malus domestica Borkh) on cv. M.7a root-stocks were assessed for the presence of fungal strands of Phymatotrichopsis omnivora (Duggar) Hennebert in two orchards in central Texas. Fungal advance within each orchard was assessed in five directions. Pathogen growth (P < 0.01) occurred beyond symptomatic trees along and perpendicularly across rows. In one orchard, 80% of the first asymptomatic trees were infected along rows, followed by 60% infection perpendicularly across rows. In the other orchard, there was 100% infection of the first asymptomatic trees along rows and 60% infection perpendicularly across rows. No growth was observed diagonally across rows in either orchard. Infrared readings of canopy temperature and differences between canopy temperature and air temperature were significant (P < 0.01) for predicting infection of asymptomatic, infected trees in one orchard. Trees were shown to have extensive taproot decay and infection of lateral roots before canopy symptoms began to develop. Root diameter appeared to have no effect on the growth of the fungus.

The role of an extracellular chitinase from Trichoderma virens Gv29-8 in the biocontrol of Rhizoctonia solani.

The role of extracellular chitinase in the biocontrol activity of Trichoderma virens was examined using genetically manipulated strains of this fungus. The T. virens strains in which the chitinase gene (cht42) was disrupted (KO) or constitutively over-expressed (COE) were constructed through genetic transformation. The resulting transformants were stable and showed patterns similar to the wild-type (WT) strain with respect to growth rate, sporulation, antibiotic production, colonization efficiency on cotton roots and growth/survival in soil. Biocontrol activity of the KO and COE strains were significantly decreased and enhanced, respectively against cotton seedling disease incited by Rhizoctonia solani when compared with the WT strain.

The arg2 gene of Trichoderma virens: cloning and development of a homologous transformation system.

The arg2 gene which encodes the small subunit of carbamoyl phosphate synthetase for Trichoderma virens has been cloned and used to develop a homologous transformation system. A genomic clone containing the arg2 gene was isolated from a cosmid library of T. virens based on complementation of an arginine auxotrophic mutant of this fungus. The predicted amino acid sequence of the arg2 gene shows 56-82% identity with homologous polypeptides from other fungi. It also contains an upstream open reading frame which encodes 24 amino acids. As is observed with other gene sequences encoding this polypeptide in filamentous fungi, the N-terminus of the predicted polypeptide showed characteristic features of a mitochondrial signal sequence. The arg2 gene was used for genetic transformation of T. virens in frequencies of up to 370 transformants/microgram of DNA. Heat-shock treatment of T. virens protoplasts increased the transformation frequency by fivefold, but more than 85% of the transformants were abortive. Both single-copy, homologous integration events and ectopic, non-homologous integration events were detected by Southern analyses of genomic DNA from transformed strains.

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens.

The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans down-stream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.

Seasonal Dynamics of Bacterial Colonization of Cotton Fiber and Effects of Moisture on Growth of Bacteria within the Cotton Boll.

A highly replicated 3-year field study was conducted to determine the seasonal patterns of bacterial colonization of cotton fiber from the time of dehiscence of the bolls (the point at which the bolls just begin to open) through harvest and commercial ginning. Bacterial numbers on fiber samples from 16 plots were determined by dilution pour plating with tryptic soy agar containing cycloheximide, and numbers of gram-negative bacteria were determined by plating on tryptic soy agar containing vancomycin and cycloheximide. Populations of bacteria varied from year to year, but in all three seasons the pattern of colonization was generally a pattern consisting of a rapid increase following opening of the bolls and a more or less stable number thereafter throughout the growing season. Gram-negative bacteria accounted for 50% or more of the recoverable bacterial population. We hypothesized that the luxuriant bacterial flora developed as a result of the availability of sufficient free water in the bolls to allow bacterial proliferation with the carbon sources remaining after fiber maturation. Therefore, laboratory experiments were conducted to determine the threshold moisture level allowing growth of bacteria on fiber in the bolls. Bacterial proliferation occurred when as little as 2% moisture was added to air-dried fiber. Using simulated bolls, we demonstrated bacterial growth resulting from dew formation on fiber held in controlled-humidity chambers.

Inoculum dynamics ofGliocladium virens associated with roots of cotton seedlings.

A system was developed to evaluate the effects of root growth of cotton seedlings on the inoculum dynamics ofGliocladium virens in nonsterile soil. In soil infested withG. virens, inoculum densities of the fungus increased when plants remained alive. After 30 days, shoots were excised and the roots allowed to deteriorate. During this portion of the experiment (30-60 days) soil inoculum densities ofG. virens declined. In infested soil without a seedling, inoculum densities remained constant throughout the duration of the experiments. Colonization of roots byG. virens was found to increase throughout the duration of the experiments. At 60 daysG. virens was recovered from approximately 60% of the root pieces (1-cm) sampled. The percentage of primary, secondary, or tertiary roots colonized was different (P = 0.01), but the total colonization of roots at three depths (0-10, 10-20, and 20-30 cm) was not different (P = 0.64). In noninfested soil, colonization of roots by indigenous propagules ofG. virens was never greater than 3%.

Cotton fleahopper and associated microorganisms as components in the production of stress ethylene by cotton.

Excised cotton terminal buds incubated with adults or nymphs of the cotton fleahopper (CFH), Pseudatomoscelis seriatus (Reuter), produced ethylene at theoretical abscission-inducing rates by 24 h after introduction of the insect. Inoculation of cotton shoot tips with three microorganisms commonly associated with CFH and cotton in all cases promoted ethylene production to theoretical abscission-inducing rates by 24 h after inoculation. CFH alone or injection of microorganisms consistently caused cotton shoot tips to darken and become soft. These changes paralleled the rise in ethylene production and did not occur in control shoot tips. Of the three microorganisms, Xanthomonas campestris pv malvacearum (Smith) Dye (XCM) produced little ethylene when grown in culture, while the two fungi, Penicillium purpurogenum Stoll and P. glabrum (Wehmer) Westling, produced higher levels. The parallel between plant response to CFH, XCM, and CFH + XCM suggests a similar mechanism of ethylene induction by these two stress agents. Since a portion of the CFH were devoid of microorganisms, yet their impact on ethylene production by cotton tissue was uniform, we propose that the primary mechanism of ethylene induction involves the insect's salivary fluids which contain cell wall hydrolyzing enzymes.

Effect of Quadrat and Core Sizes on Determining the Spatial Pattern of Criconemella sphaerocephalus.

An unmanaged pasture was sampled on four occasions (A, B, C, D) with five different quadrat sizes for Criconemella sphaerocephalus by removing a constant soil core volume of 75 cm(3) (A) and 300 cm(3) (C) from increasing quadrat areas of 0.5-8 m(2), and removing soil core volumes of increasing size - 75-1,200 cm(3) (B) and 300-4,800 cm(3) (D) - proportionally with an increase in quadrat area (0.5-8 m(2)). Frequency counts of C. sphaerocephalus were fitted to six probability distributions. The index of aggregation (b) for Taylor's power law and Morisita's index of dispersion were also calculated where appropriate. Twelve of nineteen of the sampling combinations were best described by negative binomial distribution (P = 0.05). Criconemella sphaerocephalus appeared more highly aggregated when sampled with constant soil core volumes (A and C) than from increasing soil core volumes (B and D) based on Taylor's index of aggregation (b). Morisita's index of dispersion indicated aggregation at the smallest quadrat area (0.5 m(2)) for all sampling occasions (A, B, C, D).

Positional variation in phylloplane microbial populations within an apple tree canopy.

Variation in density of epiphytic yeasts, filamentous fungi, and bacteria on apple leaves collected from eight trees at nine dates for two seasons was determined with respect to three positional factors: height, compass direction from the center of the tree, and lateral proximity to the canopy periphery. Univariate analyses of variance were performed on each of the microbial classes for each date according to a model that excluded tree effect but accounted for the positional factors with interactions. The assumption of no tree effect was explored by residual analysis and examination of the seasonal pattern of microbial densities for each tree. No persuasive evidence was obtained to invalidate this assumption. For filamentous fungi and yeasts, height and lateral position were the most significant factors withp<0.05 for yeasts at several periods. The two factors appeared to be of equal importance. Trends were less clear for bacteria, but all three positional factors and some two-way interactions seemed of some importance. For filamentous fungi and bacteria, frequently no factors were significant at a level of 0.10, but at almost all sampling dates certain positional factors and interactions were significant at a level of 0.25. Inspection of partial correlation coefficients indicated no apparent linear association between densities of most pairs of microbial classes. Implications of these results for experimental design and for the microbial ecology of the phylloplane community are discussed.

The effects of a pesticide program on microbial populations from apple leaf litter.

The leaf litter microbial community was quantitatively and qualitatively changed when a standard pesticide schedule that comprised an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees in the summer. Effects were observed for two winters by four indirect assays and three direct methods. Populations were altered qualitatively both years, but the most striking difference was the quantitative impact from year to year. Bacteria, filamentous fungi, and yeasts from treated leaves were reduced 10- to 10 000-fold between November 1976 and April 1977 and did not recover until snow cover had melted in March. Reductions in 1977-1978 were negligible. The marked seasonal difference is attributed to meteorological influences. Fluorescent pseudomonads were among the bacteria depressed by chemicals. Of the 49 genera of fungi and yeasts isolated, Coniothyrium sp., Penicillium spp., Arthrobotrys spp., and Nodulisporium sp. were appreciably reduced, whereas Typhula spp., Pleurophomella sp., Sporobolomyces spp., and Rhodotorula spp. were substantially enhanced by the spray program.

The effects of a pesticide program on non-target epiphytic microbial populations of apple leaves.

The epiphytic microbial community was quantitatively and qualitatively altered when a standard pesticide schedule that comprised applications of an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees. Effects on non-target organisms were observed for two seasons by three indirect methods and three direct methods: plating of leaf washings, imprinting of leaves onto five different media, spore fall patterns, light microscopy, scanning electron microscopy, and isolation of propagules from leaves incubated in humidity chambers. Magnitude of reduction of bacteria, filamentous fungi, yeasts, and actinomycetes varied annually and between categories of microflora. Populations from treated leaves were reduced 10- to 1000-fold in 1976 and up to 50-fold in 1977. Qualitatively, fluorescent pseudomonads and lactic acid-type bacteria were among those depressed by pesticide. Fungal populations on treated leaves were less diverse than on control leaves. Aureobasidium was only slightly affected and incidence of Sporobolomyces was substantially higher on treated leaves than on controls. The results suggest that numbers of antagonists to foliar pathogens of apple which may occur as part of the natural epiphytic microbial community may be reduced by current pesticide programs and hence have possible implications for the development of biological approaches to integrated control strategies.