John M. Whipps

Phyllosphere microbiology with special reference to diversity and plant genotype

The phyllosphere represents the habitat provided by the aboveground parts of plants, and on a global scale supports a large and complex microbial community. Microbial interactions in the phyllosphere can affect the fitness of plants in natural communities, the productivity of agricultural crops, and the safety of horticultural produce for human consumption. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonists, which is influenced by numerous environmental factors in addition to leaf physico‐chemical properties. The recent use of culture‐independent techniques has demonstrated considerable previously unrecognized diversity in phyllosphere bacterial communities. Furthermore, there is significant recent evidence that plant genotype can play a major role in determining the structure of phyllosphere microbial communities. The main aims of this review are: (i) to discuss the diversity of phyllosphere microbial populations; (ii) to consider the processes by which microbes colonize the phyllosphere; (iii) to address the leaf characteristics and environmental factors that determine the survival and growth of colonists; (iv) to discuss microbial adaptations that allow establishment in the phyllosphere habitat and (v) to evaluate evidence for plant genotypic control of phyllosphere communities. Finally, we suggest approaches and priority areas for future research on phyllosphere microbiology.

The use of colony development for the characterization of bacterial communities in soil and on roots

A simple agar plating method for the description of microbial communities is described. This method is based on the quantification of the numbers of bacterial colonies in 6–7 age-based classes as they appear on agar media over a period of 6–10 days. The method can be used to quantify microbial communities in different habitats (roots and soil) and can be related to the ecophysiology of the microbial communities present. Significant differences in distribution patterns were found in time and depth on the roots. In general, as roots matured, the microbial communities changed from one dominated by r-strategists to one that was more distributed towards K-strategists. The soil had the greatest percentage of organisms that could be characterized as K-strategists. The method was also used to compare microbial communities on wheat roots and in soil in both the field and in microcosms in the glasshouse. In general, the method enabled differentiation between r- and K-strategists in environmental samples, something that could not be done using an ecophysiological index (a modification of the Shannon diversity index) or total bacterial numbers alone.

Both Leaf Properties and Microbe-Microbe Interactions Influence Within-Species Variation in Bacterial Population Diversity and Structure in the Lettuce (Lactuca Species) Phyllosphere

ABSTRACT Morphological and chemical differences between plant genera influence phyllosphere microbial populations, but the factors driving within-species variation in phyllosphere populations are poorly understood. Twenty-six lettuce accessions were used to investigate factors controlling within-species variation in phyllosphere bacterial populations. Morphological and physiochemical characteristics of the plants were compared, and bacterial community structure and diversity were investigated using terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA gene clone libraries. Plant morphology and levels of soluble carbohydrates, calcium, and phenolic compounds (which have long been associated with plant responses to biotic stress) were found to significantly influence bacterial community structure. Clone libraries from three representative accessions were found to be significantly different in terms of both sequence differences and the bacterial genera represented. All three libraries were dominated by Pseudomonas species and the Enterobacteriaceae family. Significant differences in the relative proportions of genera in the Enterobacteriaceae were detected between lettuce accessions. Two such genera (Erwinia and Enterobacter) showed significant variation between the accessions and revealed microbe-microbe interactions. We conclude that both leaf surface properties and microbial interactions are important in determining the structure and diversity of the phyllosphere bacterial community.

Potential of Trichoderma spp. as consistent plant growth stimulators

In a series of repeated trials, six Trichoderma spp. strains, applied as a dried powder from a liquid fermentation in molasses/yeast medium, proved to be consistent at promoting the growth of lettuce (Latuca sativa L.) seedlings grown in a peat-sand potting compost in the glasshouse. Strains WT, 92, 20, and 75 at 0.75% or 1% w:w concentrations increased shoot dry weight by up to 26%, although WT did inhibit germination. For example, after 4 days only 13% of seeds sown in WT 1% w:w treated compost had germinated, whereas in other treatments germination was consistently greater than 32%. WT increased shoot fresh and dry weights by 14.3 g and 0.6 g per pot, respectively, without affecting the root dry weights, to give concomitant increases in shoot: root ratios of fresh and dry weight. The potential use of these Trichoderma spp. strains for plant growth promotion is discussed.

The tomato powdery mildew fungus Oidium neolycopersici.

UNLABELLED summary Pathogen: Powdery mildew fungus; Ascomycete although sexual stage is yet to be found; an obligate biotroph. IDENTIFICATION Superficial mycelium with hyaline hyphae; unbranched erect conidiophores; conidia, ellipsoid-ovoid or doliform, 22-46 x 10-20 microm, lack fibrosin bodies; conidia formed singly, rarely in short chains of 2-6 conidia; appressoria lobed to multilobed, rarely nipple-shaped. Pseudoidium species. HOST RANGE Broad, reported to attack over 60 species in 13 plant families, particularly members of the Solanaceae and Curcubitaceae. SYMPTOMS Powdery white lesions on all aerial plant parts except the fruit. In severe outbreaks the lesions coalesce and disease is debilitating. Agronomic importance: Extremely common in glasshouse tomatoes world wide but increasing in importance on field grown tomato crops. CONTROL Chemical control and breeding programmes for disease resistance.

Biological control of Pythium species

Rapid germination of sporangia of Pythium species in response to seed or root exudates followed by immediate infection, and the ability to cause long‐term root rots make biological control of these pathogens very difficult. Pythium‐suppressive soils exist and these may be good sources of suitable biological control agents. Both bacterial and fungal antagonists are known to affect Pythium species by producing antibiotics, competing for space or nutrients or by direct parasitism. Antagonists have been incorporated into soil or applied to seeds, and, in some instances, control of damping‐off, equivalent to standard fungicide applications, has been achieved. However, reproducible cost‐effective biological control in the field is rare. Nevertheless Pseudomonas fluorescens, Streptomyces griseoviridis, Gliocladium virens, Pythium oligandrum and Trichoderma harzianum have been or are about to be used commercially for the control of diseases caused by Pythium. The problems associated with the commercial acceptance...

Potential for Integrated Control of Sclerotinia sclerotiorum in Glasshouse Lettuce Using Coniothyrium minitans and Reduced Fungicide Application

ABSTRACT All pesticides used in United Kingdom glasshouse lettuce production (six fungicides, four insecticides, and one herbicide) were evaluated for their effects on Coniothyrium minitans mycelial growth and spore germination in vitro agar plate tests. Only the fungicides had a significant effect with all three strains of C. minitans tested, being highly sensitive to iprodione (50% effective concentration [EC(50)] 7 to 18 mug a.i. ml(-1)), moderately sensitive to thiram (EC(50) 52 to 106 mug a.i. ml(-1)), but less sensitive to the remaining fungicides (EC(50) over 200 mug a.i. ml(-1)). Subsequently, all pesticides were assessed for their effect on the ability of C. minitans applied as a solid substrate inoculum to infect sclerotia of Sclerotinia sclerotiorum in soil tray tests. Despite weekly applications of pesticides at twice their recommended concentrations, C. minitans survived in the soil and infected sclerotia equally in all pesticide-treated and untreated control soil trays. This demonstrated the importance of assessing pesticide compatibility in environmentally relevant tests. Based on these results, solid substrate inoculum of a standard and an iprodione-tolerant strain of C. minitans were applied individually to S. sclerotiorum-infested soil in a glasshouse before planting lettuce crops. The effect of a single spray application of iprodione on disease control in the C. minitans treatments was assessed. Disease caused by S. sclerotiorum was significantly reduced by C. minitans and was enhanced by a single application of iprodione, regardless of whether the biocontrol agent was iprodione-tolerant. In a second experiment, disease control achieved by a combination of C. minitans and a single application of iprodione was shown to be equivalent to that of prophylactic sprays with iprodione every 2 weeks. The fungicide did not affect the ability of C. minitans to spread into plots where only the fungicide was applied and to infect sclerotia. These results indicate that integrated control of S. sclerotiorum with soil applications of C. minitans and reduced foliar iprodione applications was feasible, did not require a fungicide tolerant isolate, and that suppression of Sclerotinia disease by C. minitans under existing chemical control regimes has credence.

Ascospore release and survival in Sclerotinia sclerotiorum

The release and survival of ascospores of a UK Sclerotinia sclerotiorum isolate were studied. Apothecia placed in a spore clock apparatus with different lighting regimes at 15 degrees C released ascospores continuously with an increasing rate for the duration of experiments (72-84 h). Spore release was not confined to light or dark periods in alternating regimes and occurred in continuous dark or light. Ascospores were released in both saturated air (90-95% rh) and at 65-75% rh. High temperature and rh were detrimental to ascospore survival but spore viability was maintained for longer periods than previously reported. The significance of these results in relation to disease control is discussed.

Characterisation of bacteria from Pinus sylvestris–Suillus luteus mycorrhizas and their effects on root–fungus interactions and plant growth

Bacteria from Pinus sylvestris-Suillus luteus mycorrhizas were isolated, characterised, and their effects on P. sylvestris-S. luteus interactions and plant growth investigated in vitro. The isolates formed five distinct phenotypic and physiological groups. Two of the groups, accounting for 34 of the 55 isolates, consisted of Bacillus spp., with three subgroups represented. The other groups contained Burkholderia spp., Serratia spp. and Pseudomonas spp. Representatives from each bacterial group were used in microcosm experiments to investigate bacterial effects on P. sylvestris-S. luteus interactions. Most Bacillus isolates stimulated growth of S. luteus along the P. sylvestris root, while isolates of Pseudomonas and Serratia inhibited root colonisation by the fungus. Burkholderia and Serratia isolates inhibited ectomycorrhiza formation by 97 and 41% respectively, while a single Bacillus isolate doubled the formation of first order ectomycorrhizal roots. There were no clear relationships between effects of the bacteria on root colonisation by the fungus after 4 weeks, and chitinase production or subsequent ectomycorrhiza formation. However, isolates that inhibited ectomycorrhiza formation appeared to associate preferentially with ectomycorrhizal roots. Several isolates enhanced plant growth substantially, although these effects were unrelated to either root colonisation by the fungus or ectomycorrhiza formation.

Plasmid Transfer between the Bacillus thuringiensis Subspecies kurstaki and tenebrionis in Laboratory Culture and Soil and in Lepidopteran and Coleopteran Larvae

ABSTRACT Plasmid transfer between Bacillus thuringiensis subsp.kurstaki HD1 and B. thuringiensis subsp.tenebrionis donor strains and a streptomycin-resistantB. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37°C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10−1 transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When aB. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp.tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.