Differentiation of Phytophthora infestans sporangia from other airborne biological particles by flow cytometry.

The ability of two different flow cytometers, the Microcyte (Optoflow) and the PAS-III (Partec), to differentiate sporangia of the late-blight pathogen Phytophthora infestans from other potential airborne particles was compared. With the PAS-III, light scatter and intrinsic fluorescence parameters could be used to differentiate sporangia from conidia of Alternaria or Botrytis spp., rust urediniospores, and pollen of grasses and plantain. Differentiation between P. infestans sporangia and powdery mildew conidia was not possible by these two methods but, when combined with analytical rules evolved by genetic programming methods, could be achieved after staining with the fluorescent brightener Calcofluor white M2R. The potential application of these techniques to the prediction of late-blight epiphytotics in the field is discussed.     

Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia

The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m⁻³ for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m⁻³ to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.     

Polymorphisms in Phytophthora infestans: Four Mitochondrial Haplotypes Are Detected after PCR Amplification of DNA from Pure Cultures or from Host Lesions

Four pairs of primers were designed for PCR amplification of known polymorphic regions of the mitochondrial genome of Phytophthora infestans. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with MspI uniquely identifies haplotypes Ib and IIa, while types Ia and IIb are differentiated by digestion of product P4 with EcoRI. Digestion of products P1 and P3 gave results similar to that with digestion of P4, but amplification of these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4. Identification of haplotypes was also possible from DNA extracted directly from small, late-blight lesions on both tomato and potato leaves, making isolation of the fungus unnecessary. A rapid and efficient method of monitoring changes in the pathogen population is facilitated. These PCR primers were also useful for differentiating other Phytophthora species.     

A Phytophthora infestans G-protein beta subunit is involved in sporangium formation

The heterotrimeric G-protein pathway regulates cellular responses to a wide range of extracellular signals in virtually all eukaryotes. It also controls various developmental processes in the oomycete plant pathogen Phytophthora infestans, as was concluded from previous studies on the role of the G-protein α-subunit PiGPA1 in this organism. The expression of the P. infestans G-protein β-subunit gene Pigpb1 was induced in nutrient-starved mycelium before the onset of sporangium formation. The gene was hardly expressed in mycelium incubated in rich growth medium. The introduction of additional copies of Pigpb1 into the genome led to silencing of the gene and resulted in transformants deficient in PiGPB1. These Pigpb1-silenced mutants formed very few asexual spores (sporangia) when cultured in rye sucrose medium and produced a denser mat of aerial mycelium than the wild type. Partially Pigpb1-silenced mutants showed intermediate phenotypes with regard to sporulation, and a relatively large number of their sporangia were malformed. The results show that PiGPB1 is important for vegetative growth and sporulation and, therefore, for the pathogenicity of this organism.     

Late blight on potato is suppressed by the biosurfactant-producing strain Pseudomonas koreensis 2.74 and its biosurfactant

Potato late blight disease caused by the zoospore-producing pathogen Phytophthora infestans (Mont.) de Bary is one of the most destructive plant diseases world-wide and currently its management mainly relies on the frequent use of fungicides. This study investigated the possibility of reducing potato late blight by biocontrol with the biosurfactant-producing strain Pseudomonas koreensis 2.74. Significant disease reduction with the biosurfactant-producing strain and its biosurfactant was observed in greenhouse trials using a detached-leaf assay. A direct effect of the biosurfactant on zoospores of P. infestans was also observed, whereas the biosurfactant only caused a minor reduction in mycelial growth rate and had no effect on the rate of sporangia production in pure culture.     

Association of melanin content with conidiogenesis in Bipolaris Sorokiniana of barley (Hordeum vulgare L.)

Sixty-seven isolates of Bipolaris sorokiniana of barley, belonging to three groups (black, white and mixed) were studied to find an association of melanin with the spore production of the fungus. Conidiogenesis in black, white and mixed subpopulation of B. sorokiniana was positively correlated with melanin content/g of mycelium. Primary hyphae of black and mixed subpopulation differentiated into secondary hyphal structures which subsequently produced conidiophores and conidia. Primary hyphae could not differentiate into secondary hyphae and subsequently conidiophores and conidia in white subpopulation. A melanin containing mutant developed from white subpopulation regained its ability to differentiate into secondary hyphae, conidiophores and conidia. Results showed that melanization of mycelia B. sorokiniana mycelia is an important factor for conidia production.     

Two new species,Pythium agreste and P. wuhanense,based on morphological characteristics and DNA sequence data

In an investigation of Pythium species in China, two new species, P. agreste and P. wuhanense, were identified based on morphological characteristics and DNA sequence data. Pythium agreste has slightly inflated sporangia, oogonia encompassed by antheridia and antheridial stalks forming a very complicated knot, and plerotic oospores. It differs from the morphologically similar P. volutum which has inflated sporangia, bigger oogonia, and aplerotic oospores; from P. kashmirense which has contiguous inflated sporangia, catenulate oogonia and coiled or bent oogonial stalks; and from P. pectinolyticum which has catenulate oogonia and bigger oogonia and oospores. Pythium wuhanense can be differentiated from morphologically similar P. emineosum and P. paroecandrum by its possession of intercalary cylindrical to elongated sporangia and intercalary oogonia catenulate with sporangia and antheridia. Phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species, based on the internal transcribed spacer (ITS) region and cytochrome C oxidase subunit I (COI) gene sequences using maximum parsimony and Bayesian methods. The two new species are described and illustrated in detail.     

Pathogen virulence of Phytophthora infestans: from gene to functional genomics

The oomycete, Phytophthora infestans, is one of the most important plant pathogens worldwide. Much of the pathogenic success of P. infestans, the potato late blight agent, relies on its ability to generate large amounts of sporangia from mycelia, which release zoospores that encyst and form infection structures. Until recently, little was known about the molecular basis of oomycete pathogenicity by the avirulence molecules that are perceived by host defenses. To understand the molecular mechanisms interplay in the pathogen and host interactions, knowledge of the genome structure was most important, which is available now after genome sequencing. The mechanism of biotrophic interaction between potato and P. infestans could be determined by understanding the effector biology of the pathogen, which is until now poorly understood. The recent availability of oomycete genome will help in understanding of the signal transduction pathways followed by apoplastic and cytoplasmic effectors for translocation into host cell. Finally based on genomics, novel strategies could be developed for effective management of the crop losses due to the late blight disease.     

Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein

The expression patterns of plant defense genes encoding osmotin and osmotin-like proteins imply a dual function in osmotic stress and plant pathogen defense. We have produced transgenic potato (Solanum commersonii Dun.) plants constitutively expressing sense or antisense RNAs from chimeric gene constructs consisting of the cauliflower mosaic virus 35S promoter and a cDNA (pA13) for an osmotin-like protein. Transgenic potato plants expressing high levels of the pA13 osmotin-like protein showed an increased tolerance to the late-blight fungus Phytophthora infestans at various phases of infection, with a greater resistance at an early phase of fungal infection. There was a decrease in the accumulation of osmotin-like mRNAs and proteins when antisense transformants were challenged by fungal infection, although the antisense transformants did not exhibit any alterations in disease susceptibility. Expression of pA13 sense and antisense RNAs had no effect on the development of freezing tolerance in transgenic plants when assayed under a variety of conditions including treatments with abscisic acid or low temperature. These results provide evidence of antifungal activity for a potato osmotin-like protein against the fungus P. infestans, but do not indicate that pA13 osmotin-like protein is a major determinant of freezing tolerance.     

Factors involved in resistance to blight (Phytophthora infestans (Mont.) de Bary) in potatoes and assessment of resistance using detached leaves

In leaves of potatoes inoculated with Phytophthora infestans early in the growing season, more but smaller lesions with later onset of sporulation and fewer sporangia developed than on those inoculated later. None of these factors was uniform on detached leaves of all clones, so could not be used separately to forecast field resistance satisfactorily; when considered together, a reasonable prediction was feasible. With some clones, tests of detached leaflets gave a better indication of behaviour in the field than laboratory tests of whole plants.     

Morphology and molecular taxonomy of Evlachovaea-like fungi,and the status of this unusual conidial genus

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.     

Improving the use of aerobiological and phenoclimatological data to forecast the risk of late blight in a potato crop

The paper describes phenological and aerobiological monitoring conducted during 6 years on a potato crop. The progression of the phenological stages in relation with thermal time [growing degree-days (GDD) and physiological days (P-days)] was analyzed. The growing cycle for Kennebec variety required less than 120 days and mean values of 1700 GDD and 720 P-days to complete the phenological development. The presence of Phytophthora infestans in the environment was common in each crop cycle, but the maximum peaks of sporangia were detected a few days after the emergence of the plants. The growing cycles with lower maximum temperature had the highest presence of sporangia. Thus, significant negative correlations between mean temperature, maximum temperature, accumulated growing degree-days, accumulated physiological days and the concentration of P. infestans in the environment were found. Maximum temperature and accumulated growing degree-days of 5 previous days were the parameters that best estimated the P. Infestans concentration in the regression model applied, with an explained variance of the data of 33 and 34%.     

Genetic Analyses of Interspecific Hybrids between Phytophthora infestans and Phytophthora mirabilis

Goodwin, S. B., and Fry, W. E. 1994. Genetic analyses of interspecific hybrids between Phytophthora infestans and Phytophthora mirabilis. Experimental Mycology 18, 20-32. Four crosses were made between isolates of two host-specific Phytophthora species. Phytophthora infestans and Phytophthora mirabilis. In the two most successful crosses involving a common P. infestans A2 parent, allozyme analysis confirmed that 79 of 86 progeny were interspecific hybrids, 3 were presumed selfs, and 4 were either selfs or nonrecombinant parental types. Mating type, alleles at the allozyme locus glucose-6-phosphate isomerase, and the + alleles at a number of DNA fingerprinting loci segregated independently according to Mendelian expectation. Three DNA fingerprinting loci were tightly linked in P. mirabilis, but no other linkages were detected among these markers. Mitochondrial DNA was uniparentally inherited, mostly from the P. infestans parent. Growth rate segregated as a quantitative character. None of the 68 progeny tested infected Mirabilis jalapa (the host of P. mirabilis), 3 infected potato, and 4 were weakly pathogenic to tomato. Because most of the F₁ hybrids could not infect any of the hosts infected by the parents, host specialization could provide a postzygotic as well as a prezygotic reproductive isolating mechanism for P. infestans and P. mirabilis in central Mexico. These results indicate that P. mirabilis probably is capable of a regular outcrossing mating system.     

Articulospora – Phylogeny vs morphology

The taxonomy of the aquatic hyphomycete genus Articulospora (Ascomycota, Pezizomycotima, Leotiales, Helotiaceae) is based on the morphology of the generative phase of its lifecycle. The type species is Articulospora tetracladia, which is distributed worldwide. Its most frequent populations in nature have dimorphic conidia, differing by the extent of conidial branching (i.e., one or two levels of branching). Some strains, stable in culture, produce exclusively conidia of one type. With the molecular analyses employed here and the relatively low number of available isolates (20), separation based on branching of conidia has not been fully supported. Therefore we propose to retain the broad concept of A. tetracladia with dimorphic conidia. Among the three gene sequences tested as potential barcodes, the internal transcribed spacer (ITS) gene was the most promising region. All strains yielded amplifiable DNA which provided adequate resolution, according to accepted ranges in inter/intraspecific differences, to differentiate among the three Articulospora and two Fontanospora species that were tested (Articulospora atra, Articulospora proliferata, A. tetracladia, Fontanospora eccentrica, Fontanospora fusiramosa). D1/D2 primers also permitted amplification in all strains, however without much resolution. Amplification of the COX1 gene sequence was least consistent.     

Monitoring airborne concentrations of sporangia ofPhytophthora infestans in relation to tomato late blight in Emilia Romagna,Italy

Late blight caused byPhytophthora infestans is one of the most important diseases of tomato crops in Emilia Romagna. In total, 7–8 chemical sprays are applied per season to control the disease without taking into account the risk of infection. An integrated warning service for the prediction of tomato late blight was set up in order to define a more rational disease control strategy. As a part of the warning service, a number of sites monitoringPh. infestans airborne sporangia was set up for a timely disease prediction in the regional tomato growing areas. The results of 3 years monitoring ofPh. infestans sporangia are presented. They suggest that concentrations of airborne sporangia rapidly increase 1–2 weeks prior the blight onset in the field.     

Late-Blight-Resistant Tomato Plants Obtained by T-DNA Insertion Mutagenesis

T-DNA insertion mutagenesis, a novel molecular-genetic approach, was used to obtain tomato (Lycopersicon esculentumMill.) plants resistant to late-blight disease caused by Phytophthora infestans(Mont.) De Bary. Plants were inoculated with a mixed pathogen population combining isolates from several sites in the Leningrad oblast. Among the plants of the primary transformant and first generation, we discerned forms with a modified leaf pattern and relative resistance to late blight. The plants with the modified leaf pattern were sterile: their bud initials never developed into flowers. The resistant plants were obtained at a frequency of 0.1%.