Marc Bardin

Microbial Biodiversity: Approaches to Experimental Design and Hypothesis Testing in Primary Scientific Literature from 1975 to 1999

SUMMARY Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

Identification of two powdery mildew fungi, Oidium neolycopersici sp. nov. and O. lycopersici , infecting tomato in different parts of the world

A world-wide study of the Oidium species causing economic damage on tomato has identified two taxa using classical morphological, scanning electron microscope (SEM) and molecular phylogenetic analyses. The material consisted of a total of 25 tomato powdery mildew isolates and 29 herbarium specimens coming from all continents where tomatoes are grown. A taxon with non-catenate conidia widespread in Europe, Africa, North and South America and Asia was identified as an O. subgen. Pseudoidium species (teleomorph: Erysiphe sect. Erysiphe). Formerly mistaken for O. lycopersicum (or O. lycopersici), it is now recognised as a distinct species, O. neolycopersici sp. nov. A phylogenetic analysis of the internal transcribed spacer (ITS) sequences of the ribosomal DNA (rDNA) indicated that O. neolycopersici is closely related to Erysiphe macleayae, E. aquilegiae and other Pseudoidium species. Only a taxon with catenate conidia was found on Australian specimens. This was identified as a species of O. subgen. Reticuloidium (teleomorph: Golovinomyces sp.). Phylogenetic analysis of the rDNA ITS sequences showed that this species is closely related to O. longipes infecting eggplant. Because it is most likely to be the same species as the original O. lycopersicum, which was actually first described in Australia, this is here neotypified as O. lycopersici.

Expanding the Paradigms of Plant Pathogen Life History and Evolution of Parasitic Fitness beyond Agricultural Boundaries

How do pathogens, whether they parasitize plants or animals, acquire virulence to new hosts and resistance to the arms we deploy to control disease? The significance of these questions for microbiology and for society at large can be illustrated by the recent worldwide efforts to track and limit the emergence of human transmissible strains of swine and avian influenza virus and of multidrug-resistant lines of human pathogenic bacteria, and to restrain the spread of Ug99, a strain of stem rust of wheat. Recent research in medical epidemiology has elucidated the impact of pathogen ecology in environmental reservoirs on the evolution of novel or enhanced pathogen virulence. In contrast, the evolution of virulence in plant pathogens has been investigated from a predominantly agro-centric perspective, and has focused overwhelmingly on evolutionary forces related to interactions with the primary plant host. Here, we argue that current concepts from the field of medical epidemiology regarding mechanisms that lead to acquisition of novel virulence, biocide resistance, and enhanced pathogenic fitness can serve as an important foundation for novel hypotheses about the evolution of plant pathogens. We present numerous examples of virulence traits in plant pathogenic microorganisms that also have a function in their survival and growth in nonagricultural and nonplant habitats. Based on this evidence, we make an appeal to expand concepts of the life history of plant pathogens and the drivers of pathogen evolution beyond the current agro-centric perspective.

Virulence variation and DNA polymorphism in Sphaerotheca fuliginea, causal agent of powdery mildew of cucurbits

Strains of Sphaerotheca fuliginea, one of the causal agents of powdery mildew of cucurbits, were examined for differences in virulence, mating type and DNA polymorphism. The 28 strains were chosen to be diverse according to host and geographic origin. Characterization of virulence phenotypes was based on the expression of symptoms on 4 species of cucurbits and 6 cultivars of melon. Two pathotypes, capable of attacking either cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ and squash cv. ‘Diamant’ or cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ were observed. Tests on melon cultivars revealed 3 races. In tests of sexual compatibility with reference strains, heterothallism was observed for all isolates. Frequency of the two mating types differed significantly in the population. DNA polymorphism was determined both by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8S DNA amplified by the polymerase chain reaction and by random amplified polymorphic DNA (RAPD). For any one of the 11 restriction enzymes tested all strains presented an identical pattern of ITS RFLP. RAPD analysis, using 22 primers which provided reproducible patterns, revealed a relatively low degree of polymorphism. Furthermore, cluster analysis based on RAPD data (152 markers) did not separate groups within the species S. fuliginea. No association could be found between virulence, mating type, geographical and host origin and RAPD patterns. The lack of association between phenotypic and molecular markers and the close fit to linkage equilibrium for the characters examined suggest that recombination may play a role in populations of S. fuliginea.

Features of air masses associated with the deposition of Pseudomonas syringae and Botrytis cinerea by rain and snowfall

Clarifying the role of precipitation in microbial dissemination is essential for elucidating the processes involved in disease emergence and spread. The ecology of Pseudomonas syringae and its presence throughout the water cycle makes it an excellent model to address this issue. In this study, 90 samples of freshly fallen rain and snow collected from 2005–2011 in France were analyzed for microbiological composition. The conditions favorable for dissemination of P. syringae by this precipitation were investigated by (i) estimating the physical properties and backward trajectories of the air masses associated with each precipitation event and by (ii) characterizing precipitation chemistry, and genetic and phenotypic structures of populations. A parallel study with the fungus Botrytis cinerea was also performed for comparison. Results showed that (i) the relationship of P. syringae to precipitation as a dissemination vector is not the same for snowfall and rainfall, whereas it is the same for B. cinerea and (ii) the occurrence of P. syringae in precipitation can be linked to electrical conductivity and pH of water, the trajectory of the air mass associated with the precipitation and certain physical conditions of the air mass (i.e. temperature, solar radiation exposure, distance traveled), whereas these predictions are different for B. cinerea. These results are pertinent to understanding microbial survival, emission sources and atmospheric processes and how they influence microbial dissemination.

Rapid Change in the Genetic Diversity of Botrytis cinerea Populations After the Introduction of Strains in a Tomato Glasshouse

In tomato glasshouses, the population structure of airborne inoculum of Botrytis cinerea depends on the production of endogenous inoculum on diseased plants as well as on incoming exogenous inoculum. Both types of inocula may contribute differently to the development of epidemics. Two strains of B. cinerea were introduced in each of four separate compartments of an experimental tomato glasshouse. We monitored their impact on disease development and on the genetic diversity of B. cinerea populations using microsatellite markers. The naturally occurring airborne inoculum of B. cinerea displayed a high level of genetic diversity and was rapidly displaced in the glasshouse, as isolates with microsatellite profiles identical to the introduced strains amounted to 66% of the inoculum sampled from the air 14 days after inoculation and 91% of those collected from stem lesions 60 days after inoculation. This suggested an important role of secondary inoculum in disease development, which is compatible with the hypothesis of a polycyclic development of gray mold epidemics in tomato glasshouses. In controlled-environment tests on tomatoes, a wide range of aggressiveness levels was observed, both for isolates sampled from the air and from lesions on plants. Hypotheses are proposed to explain the negligible impact of naturally incoming isolates on the epidemics observed inside the four glasshouse compartments.

Functional and structural comparison of pyrrolnitrin- and iprodione-induced modifications in the class III histidine-kinase Bos1 of Botrytis cinerea.

Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade. In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants) compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure, highlighted their involvement in signal transduction. E529 and M427 seem to be principally involved in osmotic signal transduction.

Resistance to bio-insecticides or how to enhance their sustainability: a review

After more than 70 years of chemical pesticide use, modern agriculture is increasingly using biological control products. Resistances to conventional insecticides are wide spread, while those to bio-insecticides have raised less attention, and resistance management is frequently neglected. However, a good knowledge of the limitations of a new technique often provides greater sustainability. In this review, we compile cases of resistance to widely used bio-insecticides and describe the associated resistance mechanisms. This overview shows that all widely used bio-insecticides ultimately select resistant individuals. For example, at least 27 species of insects have been described as resistant to Bacillus thuringiensis toxins. The resistance mechanisms are at least as diverse as those that are involved in resistance to chemical insecticides, some of them being common to bio-insecticides and chemical insecticides. This analysis highlights the specific properties of bio-insecticides that the scientific community should use to provide a better sustainability of these products.

Causal agents of powdery mildew of cucurbits in Sudan.

In a survey of cucurbit crops in Sudan, 234 samples of melon, cucumber, and squash showing symptoms of powdery mildew were infected with Sphaerotheca fuliginea, while Erysiphe cichoracearum was only observed on two samples of watermelon showing mild symptoms. Leveillula taurica was not observed on any sample. Field trials with differential lines of melon revealed the presence of race 1 of S. fuliginea in Gezira (Central Sudan) in the summer of 1993, and race 2 in the following winter. Virulence tests in controlled conditions on a limited number of isolates suggest that a shift in prevalence of those races may have occurred on that site over time. The geographic scale of this shift was probably limited as race 0 was observed in late autumn in northeast Sudan in the Gash Delta

Is the efficacy of biological control against plant diseases likely to be more durable than that of chemical pesticides

The durability of a control method for plant protection is defined as the persistence of its efficacy in space and time. It depends on (i) the selection pressure exerted by it on populations of plant pathogens and (ii) on the capacity of these pathogens to adapt to the control method. Erosion of effectiveness of conventional plant protection methods has been widely studied in the past. For example, apparition of resistance to chemical pesticides in plant pathogens or pests has been extensively documented. The durability of biological control has often been assumed to be higher than that of chemical control. Results concerning pest management in agricultural systems have shown that this assumption may not always be justified. Resistance of various pests to one or several toxins of Bacillus thuringiensis and apparition of resistance of the codling moth Cydia pomonella to the C. pomonella granulovirus have, for example, been described. In contrast with the situation for pests, the durability of biological control of plant diseases has hardly been studied and no scientific reports proving the loss of efficiency of biological control agents against plant pathogens in practice has been published so far. Knowledge concerning the possible erosion of effectiveness of biological control is essential to ensure a durable efficacy of biological control agents on target plant pathogens. This knowledge will result in identifying risk factors that can foster the selection of strains of plant pathogens resistant to biological control agents. It will also result in identifying types of biological control agents with lower risk of efficacy loss, i.e., modes of action of biological control agents that does not favor the selection of resistant isolates in natural populations of plant pathogens. An analysis of the scientific literature was then conducted to assess the potential for plant pathogens to become resistant to biological control agents.