Survival of plant pathogens in static piles of ground green waste.

Ground green waste is used as mulch in ornamental landscapes and for tree crops such as avocados. Survival of Armillaria mellea, Phytophthora cinnamomi, Sclerotinia sclerotiorum, and Tylenchulus semipenetrans was assessed for 8 weeks within unturned piles of either recently ground or partially composted green waste. S. sclerotiorum survived at the pile surface and at 10, 30, and 100 cm within the pile for the entire 8 weeks in both fresh green waste (FGW) and aged green waste (AGW). A. mellea and T. semipenetrans did not survive more than 2 days in FGW, while P. cinnamomi persisted for over 21 days in FGW. AGW was less effective in reducing pathogen viability than FGW, most likely because temperatures in AGW peaked at 45 degrees C compared with 70 degrees C in FGW. Survival modeling curves based on pile temperatures indicate the time to inactivate 10 propagules of pathogens was 11, 30, 363, and 50 days for A. mellea, P. cinnamomi, S. sclerotiorum, and T. semipenetrans, respectively. Sclerotia-forming pathogens pose the greatest risk for escape; to ensure eradication of persistent fungi, green waste stockpiles should be turned intermittently to mix pile contents and move pathogen propagules to a location within the pile where they are more likely to be killed by heat, microbial attack, or chemical degradation.

Repetitive Applications of the Biocontrol Agent Pseudomonas putida 06909-rif/nal and Effects on Populations of Phytophthora parasitica in Citrus Orchards.

ABSTRACT Pseudomonas putida 06909-rif/nal was applied repetitively during the irrigation season in two citrus orchards over 3 years. In a mature (50-yearold) commercial citrus orchard covering 2.02 ha, weekly applications of Pseudomonas putida 06909-rif/nal with an in-field fermentor resulted in soil populations that fluctuated between 2.83 log CFU + 1 per g of soil and 4.35 log CFU + 1 per g of soil. Resulting rhizosphere populations of Phytophthora parasitica were significantly reduced in 1999 but not 1997 or 1998. In a newly planted citrus orchard, yearly applications of Pseudomonas putida 06909-rif/nal at the beginning of the irrigation season resulted in high soil populations of Pseudomonas putida 06909-rif/nal that declined rapidly and never reduced the rhizosphere populations of Phytophthora parasitica. When Pseudomonas putida 06909-rif/nal was applied weekly, soil populations increased throughout the 1997 and 1998 irrigation seasons, reaching a maximum in 1998 and remained high throughout the 1999 irrigation season. Rhizosphere populations of Phytophthora parasitica were significantly reduced in 1998. Yearly applications of the fungicide metalaxyl and the nematicide phenamiphos reduced rhizosphere populations of Phytophthora parasitica in 1997 but not in 1998 or 1999. Pseudomonas putida 06909-rif/nal was uniformly distributed throughout the soil profile to a depth of 75 cm in both yearly and weekly applications. When applied through low-volume minisprinklers, Pseudomonas putida 06909-rif/nal was found in aerosols up to 3 m away.

Effect of Repetitive Applications of the Biocontrol Bacterium Pseudomonas putida 06909-rif/nal on Citrus Soil Microbial Communities.

ABSTRACT The effects of repetitive applications of Pseudomonas putida 06909-rif/nal on the resident microbial communities within a citrus orchard were studied with fatty acid methyl-ester (FAME) profiles and ribosomal intergenic spacer analysis. The data set from FAME was large and very complex, requiring 23 factors from principal component analysis to explain 91% of variability in the data. Spatial and temporal effects on variation within microbial communities were much greater than the effects of either yearly applications of Pseudomonas putida 06909-rif/nal, weekly repetitive applications of Pseudomonas putida 06909-rif/nal, or yearly applications of the fungicide metalaxyl and the nematicide phenamiphos. Multivariate analysis of covariance showed much of the variability between treatments could be accounted for by populations of Pseudomonas putida 06909-rif/nal. Soil fatty acids that showed significant changes between treatments were not related to fatty acids found in Pseudomonas putida 06909-rif/nal, suggesting applications of Pseudomonas putida 06909-rif/nal altered the soil microbial community.

16S rDNA fingerprinting of rhizosphere bacterial communities associated with healthy and Phytophthora infected avocado roots.

Molecular techniques employing 16S rDNA profiles generated by PCR-DGGE were used to detect changes in bacterial community structures of the rhizosphere of avocado trees during infection by Phytophthora cinnamomi and during repeated bioaugmentation with a disease suppressive fluorescent pseudomonad. When the 16S rDNA profiles were analyzed by multivariate analysis procedures, distinct microbial communities were shown to occur on healthy and infected roots. Bacterial communities from healthy roots were represented by simple DNA banding profiles, suggestive of colonization by a few predominant species, and were approximately 80% similar in structure. In contrast, roots that were infected with Phytophthora, but which did not yet show visible symptoms of disease, were colonized by much more variable bacterial communities that had significantly different community structures from those of healthy roots. Root samples from trees receiving repeated applications of the disease suppressive bacterium Pseudomonas fluorescens st. 513 were free of Phytophthora infection, and had bacterial community structures that were similar to those of nontreated healthy roots. Sequence analysis of clones generated from four predominant bands cut from the DGGE gels revealed the presence of pseudomonads, as well as several previously unidentified bacteria. Differentiation of 16S rDNA profiles for healthy and infected roots suggests that rhizosphere bacterial community structure may serve as an integrative indicator of changes in chemical and biological conditions in the plant rhizosphere during the infection process.

Evaluation of Continuous Application Technology for Delivery of the Biocontrol Agent Pseudomonas putida 06909-rif/nal.

We evaluated a commercial field fermentor as a means of culturing and delivering a bacterial biocontrol agent, Pseudomonas putida 06909-rif/nal, through irrigation water. There was no evidence of contamination in 12-hour cultures produced by the fermentor. It produced 120 liters of inoculum at 5 × 108 CFU/ml after 12-hour fermentations, allowing two applications per day. Dilutions up to 1:100,000 of inoculum of P. putida 06909-rif/nal produced by the fermentor allowed the biocontrol agent to effectively colonize soil in greenhouse trials. Bacteria produced in 19-hour fermentations colonized soil better than bacteria produced in 12-hour fermentations. Ten repetitive applications of P. putida 06909-rif/nal yielded soil population levels similar to those from a single application at 10-fold greater concentrations. Repetitive applications of lower concentrations may be a cost-effective method for delivering bacterial biocontrol agents to large acreages. This work suggests that the commercial field fermentor would be a viable instrument for delivering bacterial biocontrol agents for commercial-scale field applications.

Effects of Cellulytic Enzymes on Phytophthora cinnamomi.

ABSTRACT Two enzyme systems, cellulase (beta-1,4-glucanase) and laminarinase (beta-1,3-glucanase), were added to soil extracts to simulate (in vitro) lytic components found in mulches suppressive to Phytophthora cinnamomi. Concentration ranges of each enzyme were incubated with Phytophthora cinnamomi mycelium, zoospores, zoospores cysts, and zoospore-infected excised roots to evaluate the roles of each enzyme in potential control of avocado root rot disease. Cellulase significantly retarded the development of zoosporangia and chlamydospores when mycelia were incubated in soil extract containing the enzyme at concentrations greater than 10 units/ml. Zoospore production was also reduced by cellulase but not by laminarinase. Laminarinase had little effect on zoosporangia or chlamydospore formation. At high concentrations, laminarinase was consistently more effective at preventing encystment than cellulase. Chlamydospores preformed in root tips were immune to the lytic effects of all treatments except cellulase at 100 units/ml. Zoospores placed in enzyme solutions and plated on a selective medium survived high cellulase concentrations and formed colonies, but there were fewer surviving zoospores when laminarinase was present at greater than 10 units/ml. Low concentrations of cellulase stimulated infection of excised roots, however, low concentrations of laminarinase prevented infection. Cellulase and laminarinase have different effects on the structures of the Phytophthora cinnamomi life history, however, each enzyme may have a role in reduction of inoculum.

Association of Cellulytic Enzyme Activities in Eucalyptus Mulches with Biological Control of Phytophthora cinnamomi.

ABSTRACT A series of samples were taken from mulched and unmulched trees starting at the surface of mulch or soil to a 15 cm soil depth, forming a vertical transect. Saprophytic fungi isolated from the soil samples on rose bengal medium and surveyed visually were most abundant in mulches and at the interface of mulch and soil (P < 0.05). Microbial activity as assayed by the hydrolysis of fluorescein diacetate was significantly greater in mulch layers than in soils. Cellulase and laminarinase enzyme activities were greatest in upper mulch layers and rapidly decreased in soil layers (P < 0.05). Enzyme activities against Phytophthora cinnamomi cell walls were significantly greater in mulch than in soil layers. When Phytophthora cinnamomi was incubated in situ at the various transect depths, it was most frequently lysed at the interface between soil and mulch (P < 0.001). Roots that grew in mulch layers were significantly less infected with Phytophthora cinnamomi than roots formed in soil layers. In mulched soil, roots were commonly formed at the mulch-soil interface where Phytophthora populations were reduced, whereas roots in unmulched soil were numerous at the 7.5 cm depth where Phytophthora cinnamomi was prevalent. Enzyme activities were significantly and positively correlated with each other, microbial activity, and saprophytic fungal populations, but significantly and negatively correlated with Phytophthora recovery.

Effects of Gypsum Soil Amendments on Avocado Growth, Soil Drainage, and Resistance to Phytophthora cinnamomi.

Infection of avocado seedlings by Phytophthora cinnamomi in infested soil was decreased by 71% by the addition of gypsum soil amendments in replicated greenhouse experiments. Root weights and total seedling weights were not significantly increased by gypsum amendments compared with unamended soil; however, the significant reduction in total seedling weight and root weight caused by P. cinnamomi was largely eliminated by the addition of gypsum. Fresh organic matter amendments alone did not significantly affect the total fresh weight or root weight of avocado seedlings. Root fresh weight was decreased in uninfested soil amended with 5% gypsum and organic matter, but in infested soils, the same treatment increased root fresh weight. Root fresh weight of mature avocado trees in an uninfested grove was not significantly affected by gypsum soil amendments. Avocado seedlings grown in gypsum-amended soil and the roots inoculated with suspensions of Phytophthora cinnamomi zoospores were no more resistant than seedlings grown in unamended soil. Permeability of avocado root membranes, as determined by the amount of 86Rb exuded from root segments over time, was unaffected by growth in gypsum-amended soil. Infiltration of water into soil amended with fine-grade gypsum was impeded initially; whereas soil amended with coarse drywall gypsum drained faster than unamended soil. Drainage was not correlated with root infection of avocados grown in soil infested with P. cinnamomi and amended with gypsum. It appears that large reductions in infection of avocado seedlings by P. cinnamomi in gypsum-amended soil are not caused by an avocado growth response, increased root resistance, or reduced root membrane permeability. Infection is not markedly affected by poor drainage when the soil is amended with high levels of gypsum.

Effects of Gypsum on Zoospores and Sporangia of Phytophthora cinnamomi in Field Soil.

Sporangial production of Phytophthora cinnamomi buried in gypsum-amended avocado soil for 2 days was reduced by as much as 74% in greenhouse trials. P. cinnamomi sporangial volume was reduced an average of 64% in gypsum-amended soil. Soil extracts from gypsum-amended soil reduced in vitro sporangial production and volume. Irrigation with gypsum solutions of buried mycelium in unamended soil also reduced sporangial production and volume. Zoospore production and colony-forming units of P. cinnamomi were reduced in soil amended with calcium sulfate, calcium nitrate, or calcium carbonate. Zoospore encystment or passive movement through soil was not significantly affected by gypsum soil amendments.

First Report of Entyloma Polysporum on Sunflower (Helianthus annuus) in Southern California.

During a period of wet weather from December 1996 to March 1997, commercial plantings of sunflower in San Diego County, CA, were infected by a leaf smut, Entyloma polysporum (Peck) Farl. The fungus was observed on sunflowers grown in a greenhouse in San Diego County and also on sunflowers from nurseries in Ventura and Riverside counties. Although the disease was first noticed in 1996, the infection was not of economic significance so no attempt was made to identify the causal agent. However, with continuous cropping of sunflowers year round significant losses were observed on seedlings that were systemically infected as they emerged. This is the first report of E. polysporum causing economic losses on sunflowers. The distinguishing characteristics of this fungus are masses of globose to subglobose spores, pale green to yellow green in color, approximately 12 µm in diameter, with a double wall consisting of an inner pale green wall and outer hyaline sheath. The spores occur in dense masses called sori that completely replace the leaf cells. Young spores are difficult to distinguish from leaf cells in a cursory examination. Older sori form discolored lesions in the leaf ranging from circular to irregular in shape and replace most of the chlorenchyma tissue in the infected lesions. Identification of species of smut fungi such as Entyloma is based on the location of the sori in the vegetative parts of the host, the identification of the host, and the spore morphology (4). Savile (3) reviewed the genus on North American composites and, based on morphological characteristics, concluded that almost all the pathogens were either E. compositarum or E. polysporum, with a few intermediate forms. E. polysporum is characterized by globose spores 10 to 17 µm in diameter, surrounded by cell walls 1 µm thick encased in a 1.5- to 2.5-µm thick smooth hyaline sheath (1). Spores of E. compositarum are smaller, 9 to 12 µm in diameter, thin walled (1 to 1.5 µm), smooth, and without a sheath (2). Vánky (4) lists 33 different species on composites according to their host. He believes E. polysporum only occurs on Ambrosia spp., and does not include E. compositarum in his list of Entyloma spp. Neither author mentions Entyloma infecting any species of Helianthus. Savile concluded that E. calendulae (Oudem.) de Bary, described in Europe, is very similar morphologically to E. polysporum, and is probably the same species. E. polysporum was first described in 1881 by Peck as Protomyces polysporus infecting Ambrosia trifida in New York State. In 1996, it was described on Ambrosia artemesifolia in Hungary (3). In the United States it has been reported on sunflowers in Montana (1,2). References: (1) D. F. Farr et al. 1989. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN. (2) G. W. Fischer. 1953. Manual of the North American Smut Fungi. Ronald Press, New York. (3) D. B. O. Savile. Can. J. Res. 25(C):109, 1947. (4) K. Vánky. 1994. European Smut Fungi. Gustav Fischer, New York.

Mutations Affecting Hyphal Colonization and Pyoverdine Production in Pseudomonads Antagonistic toward Phytophthora parasitica.

In previous studies, Pseudomonas putida 06909 and Pseudomonas fluorescens 09906 suppressed populations of Phytophthora parasitica in the citrus rhizosphere, suggesting that these bacteria may be useful in biological control of citrus root rot. In this study we investigated the mechanisms of antagonism between the bacteria and the fungus. Both bacteria colonized Phytophthora hyphae and inhibited the fungus on agar media. A hyphal column assay was developed to measure the colonization of bacteria on fungal hyphae and to enrich for colonization-deficient mutants. In this way we identified Tn5 mutants of each pseudomonad that were not able to colonize the hyphae and inhibit fungal growth in vitro. Colonization-deficient mutants were nonmotile and lacked flagella. Survival of nonmotile mutants in a citrus soil was similar to survival of a random Tn5 mutant over a 52-day period. Additional screening of random Tn5 mutants of both pseudomonads for loss of fungal inhibition in vitro yielded two distinct types of mutants. Mutants of the first type were deficient in production of pyoverdines and in inhibition of the fungus in vitro, although they still colonized fungal hyphae. Mutants of the second type lacked flagella and were not able to colonize the hyphae or inhibit fungal growth. No role was found for antibiotic production by the two bacteria in the inhibition of the fungus. Our results suggest that both hyphal colonization and pyoverdine production are important in the inhibition of Phytophthora parasitica by P. fluorescens and P. putida in vitro.

Role of copper resistance in competitive survival of Pseudomonas fluorescens in soil.

A copper-resistant strain (09906) of Pseudomonas fluorescens that was isolated from a citrus grove soil is being investigated as a biological control agent for Phytophthora root rot. Since citrus grove soils in California are often contaminated with copper from many years of copper fungicide applications, the role of copper resistance in survival of strain 09906 was investigated. Three copper-sensitive Tn5 mutants were obtained with insertions in different chromosomal DNA regions. These insertions were not in the chromosomal region that hybridized with the copper resistance operon (cop) cloned from Pseudomonas syringae. A copper-sensitive mutant survived as well as the wild type in a sterile loamy sand without added copper, but with 10 and 15 micrograms of CuSO4 added per g of soil, populations of the copper-sensitive mutant were 27- and 562-fold lower, respectively, than that of the wild type after a 25-day period. In a sterilized citrus grove soil, populations of the copper-sensitive mutant and wild-type strain were similar, but in nonsterile citrus soil, populations of the copper-sensitive mutant were 112-fold lower than the wild type after 35 days. These data suggest that copper resistance genes can be important factors in persistence of P. fluorescens in soil contaminated with copper. In addition, these genes appear to play a role in competitive fitness, even in soils with a low copper content.

Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation.

A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular-arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study.

Membrane-mediated decrease in root exudation responsible for phorphorus inhibition of vesicular-arbuscular mycorrhiza formation.

The mechanism responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation in sudangrass (Sorghum vulgare Pers.) was investigated in a phosphorus-deficient sandy soil (0.5 micrograms phosphorus per gram soil) amended with increasing levels of phosphorus as superphosphate (0, 28, 56, 228 micrograms per gram soil). The root phosphorus content of 4-week-old plants was correlated with the amount of phosphorus added to the soil. Root exudation of amino acids and reducing sugars was greater for plants grown in phosphorus-deficient soil than for those grown in the phosphorus-treated soils. The increase in exudation corresponded with changes in membrane permeability of phosphorus-deficient roots, as measured by K(+) ((86)Rb) efflux, rather than with changes in root content of reducing sugars and amino acids. The roots of phosphorus-deficient plants inoculated at 4 weeks with Glomus fasciculatus were 88% infected after 9 weeks as compared to less than 25% infection in phosphorus-sufficient roots; these differences were correlated with root exudation at the time of inoculation. For plants grown in phosphorus-deficient soil, infection by vesicular-arbuscular mycorrhizae increased root phosphorus which resulted in a decrease in root membrane permeability and exudation compared to nonmycorrhizal plants. It is proposed that, under low phosphorus nutrition, increased root membrane permeability leads to net loss of metabolites at sufficient levels to sustain the germination and growth of the mycorrhizal fungus during pre- and postinfection. Subsequently, mycorrhizal infection leads to improvement of root phosphorus nutrition and a reduction in membrane-mediated loss of root metabolites.

Interaction Between Meloidogyne arenaria and Glomus fascicuqlatus in Grape.

Root zones of grape (Fitis vinifera cv Thompson Seedless) cuttings were infested with chlamydospores of Glomus fasciculatus or eggs of Meloidogyne arenaria or both. Growth of grapevines was greatest in mycorrhizal (G. fasciculatus) plants. Mycorrhizal development and growth of mycorrhizal and nonmycorrhizal plants were reduced in the presence of M. arenaria. At low initial nematode inoculum (PI) levels (approx. 200 eggs/plant), the presence of mycorrhizae enhanced plant growth during 1 yr, but no significant benefit was achieved by mycorrhizae where PI was high (approx. 2,000 eggs/plant). Final nematode populations were highest in mycorrhizal plants.