First report of Solanum physalifolium as a host plant for Phytophthora infestans in Sweden

Andersson B, Johansson M and Jönsson B. 2003. Plant Disease 87:1538.

In the early summer of 2003, lesions resembling those caused by Phytophthora infestans (Mont.) de Bary on potato were observed on Solanum physalifolium Rusby var. nitidibaccatum (Bitter) Edmonds (2) that was growing as a weed in a parsnip (Pastinaca sativa) field in sourthern Sweden. When infected leaves of S. physalifolium were observed under the microscope (x200 magnification), sporangia with the same shape and size as those of P. infestans were observed. Pieces of infected leaves of S. physalifolium were put under tuber slices of S. tuberosum (cv. Bintje) in petri dishes and kept at 20°C. After 4 days, mycelium grew through the slices and sporulated profusely. The sporangia on the slices were of the same shape and size as those observed on the infected S. physalifolium leaves. In Sweden, the ratio of A1 and A2 mating types is 50:50, and oospores are commonly found in infected potato crops (1), so isolates from S. physalifolium were tested for mating type by growing them together with reference isolates of a known mating type on agar plates. Nine of the tested isolates were A1 mating type and six were A2 mating type. One self-fertile isolate was found. Naturally infected leaves of S. physalifolium were incubated at 20°C at 100% relative humidity so the lesions could coalesce and to facilitate oospore formation. After 5 days, oospores identical to those of P. infestans were observed under the microscope (x200 magnification). Sporangia produced by isolates originating from S. physalifolium and S. tuberosum were harvested, and a suspension containing 104 sporangia from from each isolate was prepared. Five leaves each of S. nigrum, S. physalifolium and S. tuberosum (cv. Bintje), were inoculated with 10 μl of each sporangial suspension. Inoculated leaves were incubated in sealed petri dishes at 15°C. After 4 days, all S. tuberosum leaves were infected. After 7 days, two of five leaves of S. physalifolium leaves inoculated with the S. tuberosum isolate and two of five S. physalifolium leaves inoculated with the isolate from S. physalifolium were infected. All lesions produced sporangia similar to those formed by P. infestans. S. nigrum was not infected by any of the isolates. The ability of S. physalifolium to act as a host plant for P. infestans producing sporangia during the growing season and oospores for survival between growing seasons may increase the problems of controlling late blight in potato in Sweden.

References: (1) J. Dahlberg et al. Field survey of oospore formation by Phytophthora infestans. (Poster Abstr.) Pages 134-135 In: Late Blight: Managing the Global Threat. Proc Global Late Blight Conf. Charlotte Lizarraga, ed. Centro Internacional de La Papa. On-line publication, ISBN 929060-215-5, 2002. (2) J. M. Edmonds. Bot. J. Linn. Soc. 92:1. 1986

Corresponding author: Björn Andersson, Email: bjorn.andersson(at)evp.slu.se

Root and foot rot on tomato caused by Phytophthora infestans detected in Belgium

 Lievens, B, Hanssen I R M, Vanachter A  C R C, Cammue B P A and Thomma B P H J. 2004. Plant Disease 88:86.

 In January 2003, a severe root and foot rot was observed on 2-month-old wilted tomato (Lycopersicon esculentum Mill.) plants in a large-scale (2.5 ha) commercial greenhouse setting in Belgium. Tomato plants (10%) produced from healthy nursery-grown seedlings and planted to new, clean rockwool and drip irrigation with UV-disinfected water developed symptoms. Symptom development was restricted to lower plant parts with severe rotting of the entire root system and dark lesions girdling the stem base. No symptoms of disease were observed on the foliage or upper stems. Cross sections of the stem base revealed brown discoloration of internal tissue, including the vascular tissue and pith. Dark brown lesions also occurred on the roots. Sections of the stem base, the upper roots (sampled near to the stem base), and the lower roots (sampled on roots deeper in the rockwool) were plated separately on corn meal agar. The oomycete pathogen Phytophthora infestans (Mont.) de Bary was identified in each sample on the basis of morphological characteristics observed directly with light microscopy. Branched sporangiophores with slight swellings and characteristic lemon-shaped sporangia (35 x 20 µm and ratio length/width of 1.75 µm) at their tips were obvious after incubation in darkness at 24°C. Oospores and chlamydospores were not observed. After multiple soil treatment with oomycete-specific fungicides, the plants recovered. Since the occurrence of P. infestans on roots is unusual, the identity of the pathogen on the diseased plant tissues was confirmed with three techniques, DNA array identification, internal transcribed spacer (ITS) sequencing, and a polymerase chain reaction (PCR) amplification using P. infestans-specific primers. DNA was directly processed from separate samples of upper and lower root and stem base tissue. The DNA array used was originally developed to detect and identify the key fungal pathogens of tomato (2). Among detector probes for other tomato pathogens, this array contains oligonucleotide detector probes for P. infestans (PIN1: 5’-GGT TGT GGA CGC TGC TAT T and PIN2: 5’-AAT GGA GAA ATG CTC GAT TC). These probes are based on ITS sequences (ITS I and ITS II). Using conserved ribosomal primers OOMUP18Sc (5’-TGC GGA AGG ATC ATT ACC ACA C) and ITS4, oomycete DNA was amplified by PCR and simultaneously labeled with alkaline-labile digoxigenin (2). All generated amplicons strongly hybridized to the oligonucleotide detector probes for P. infestans and not to any other pathogen-specific detector probe present on the array. The pathogen could not be detected in roots and stem bases of symptomless plants. In addition, the ITS-region was sequenced and showed 100% homology with multiple GenBank accessions of P. infestans sequences. As a third confirmatory test, a PCR was performed on DNA extracts from infected root and stem base tissues using a primer set specific to P. infestans (O8-3/O8-4 [1]). A band of the expected size was produced for the infected stem base and root samples. Until now, this pathogen was known worldwide to cause late blight on potatoes and tomatoes. To our knowledge, this is the first report of root and foot rot of tomato caused by P. infestans.

 References: (1) H. S. Judelson and P. W. Tooley. Phytopathology 90:1112, 2000. (2) B. Lievens et al. FEMS Microbiol. Lett 223:113, 2003.

Corresponding author: Bart Thomma, Email: Bart.Thomma(at)wur.nl

Soilborne oospores of Phytophthora infestans in central Mexico survive winter fallow and infect potato plants in the field

 Fernández-Pavía S P, Grünwald N J, Díaz-Valasis M, Cadena-Hinojosa M and Fry W E. 2004. Plant Disease 88:29-33.

Survival and infectivity of oospores in soils naturally infested with P. infestans oospores were studied in central Mexico. Sporangia were selectively eliminated from soil samples to determine infectivity attributable to the presence of oospores. Selective elimination of sporangia was achieved by two cycles of wetting and drying the soil. Oospore concentration, viability, and infectivity varied among soils collected during the winter fallow in different locations of central Mexico. In some soils, oospores were infective regardless of the time at which they were collected during the winter fallow. However, oospore viability and infectivity decreased following 2 years of intercropping. The number of stem lesions and initial disease severity were significantly higher in soils with moderate (20 to 39 oospores g-1) soil) oospore infestation compared with soils with low (0 to 19 oospores g-1) soil) infestation. Our study confirms that oospores can survive winter fallow and serve as a source of primary inoculum in the central highlands of Mexico. Oospore survival appeared lower in the Toluca Valley soil, which may be an indication of soil suppressiveness.

 Corresponding author: Nik Grünwald, Email: ngrunwald(at)pars.ars.usda.gov

Tolerance of mycelium of different genotypes of Phytophthora infestans to freezing temperatures for extended periods

 Kirk W W. 2003. Phytopathology 93:1400-1406.

 Mycelium of Phytophthora infestans, the causal agent of potato late blight, can initiate crop infections over successive years by overwintering in infected potato tubers that survive as seed in fields or within cull piles. This study used four different genotypes of P. infestans to evaluate the influence of freezing temperatures on survival of mycelium in vitro. Sporangium-free mycelium of P. infestans US1, US8, US11, and US14 growing on rye agar plates was exposed to temperatures ranging from -20 and 0°C (experiment A) for different periods up to 24 h and from -5 and 0°C (experiment B) for periods up to 5 days. Cultures were incubated at 12°C after exposure, and survival of the cultures was estimated after 28 days by a digital image analysis technique that measured the average reflectance intensity (ARI) of images of the mycelium of temperature-treated cultures. The ARI values of treated cultures were compared with the growth of mycelium in negative controls (mycelium not present) and positive controls (mycelium exposed to 12°C for an equivalent period), and determination of recovery was based on statistical differences from the controls. There were significant differences in ARI values among genotypes, temperature treatment, and exposure periods in all experiments. An index of recovery was calculated for each genotype at all treatment temperatures and exposure periods for both experiments. In experiment A, exposure of mycelium of P. infestans (all genotypes) to -20 and -10°C proved lethal for exposure periods of more than 1 h. All genotypes showed some degree of recovery up to 24-h exposure at -5 and -3°C. In both experiments, exposure of mycelium of P. infestans to 0°C was not lethal to any genotype tested for any exposure period. In experiment B, all of the genotypes survived exposure up to 3 days at -3°C to some degree, but at -5°C, exposure of 1 day was lethal to all genotypes. Tolerance of freezing temperatures by mycelium of P. infestans may be an ecologically important survival mechanism and the increased tolerance of US8 and US14 may explain their predominance in cooler climates such as north-central United States.

Corresponding author: Willie Kirk, Email: kirkw(at)msu.edu

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

Latijnhouwers M and Govers F. 2003. Eukaryotic Cell 2: 971–977 © 2003 American Society for Microbiology

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.

Corresponding author: Francine Govers, Email: Francine.Govers(at)wur.nl

A Gα subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans

Latijnhouwers M, Ligterink W, Vleeshouwers V G A A, van West P and Govers F. 2004. Molecular Microbiology 51: 925-936. © 2003 Blackwell Publishing Ltd.

The heterotrimeric G-protein pathway is a ubiquitous eukaryotic signaling module that is known to regulate growth and differentiation in many plant pathogens. We previously identified Pigpa1, a gene encoding a G-protein α subunit from the potato late blight pathogen Phytophthora infestans. P. infestans belongs to the class oomycetes, a group of organisms in which signal transduction processes have not yet been studied at the molecular level. To elucidate the function of Pigpa1, PiGPA1-deficient mutants were obtained by homology-dependent gene silencing. The Pigpa1-silenced mutants produced zoospores that turned six to eight times more frequently, causing them to swim only short distances compared with the wild type. Attraction to the surface, a phenomenon known as negative geotaxis, was impaired in the mutant zoospores, as well as autoaggregation and chemotaxis towards glutamic and aspartic acid. Zoospore production was reduced by 20-45% in different Pigpa1-silenced mutants. Transformants expressing constitutively active forms of PiGPA1, containing amino acid substitutions (R177H and Q203L), showed no obvious phenotypic differences from the wild-type strain. Infection efficiencies on potato leaves ranged from 3% to 14% in the Pigpa1-silenced mutants, compared with 77% in wild type, showing that virulence is severely impaired. The results prove that PiGPA1 is crucial for zoospore motility and for pathogenicity in an important oomycete plant pathogen.

Corresponding author: Francine Govers, Email: Francine.Govers(at)wur.nl

Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans

Vijn I and Govers F. 2003. Molecular Plant Pathology 4:459-467. © 2003 Blackwell Publishing Ltd.

Agrobacterium tumefaciensis widely used for plant DNA transformation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late blight. Binary T-vectors containing neomycin phosphotransferase (npt) and β-glucuronidase (gus) fused to oomycete transcriptional regulatory sequences were constructed. Seven days of co-cultivation followed by transfer to a selective medium containing cefotaxim to kill Agrobacterium and geneticin to select for transformants, resulted in geneticin resistant colonies. Under optimal conditions with Agrobacterium supplemented with a ternary plasmid carrying a constitutive virG gene and in the presence of acetosyringone as inducer, up to 30 transformants per 107 zoospores could be obtained. The majority of these transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. Using a similar protocol, geneticin resistant transformants of two other other oomycetes species were obtained, Phytophthora palmivora and Pythium ultimum.

Corresponding author: Francine Govers, Email: Francine.Govers(at)wur.nl

Oomycetes and fungi: similar weaponry to attack plants

Latijnhouwers M, de Wit P J G M and Govers F. 2003. Trends in Microbiology 11: 462-469. © Elsevier Ltd.

Fungi and Oomycetes are the two most important groups of eukaryotic plant pathogens. Fungi form a separate kingdom and are evolutionarily related to animals. Oomycetes are classified in the kingdom Protoctista and are related to heterokont, biflagellate, golden-brown algae. Fundamental differences in physiology, biochemistry and genetics between fungi and Oomycetes have been described previously. These differences are also reflected in the large variations observed in sensitivity to conventional fungicides. Recently, more pronounced differences have been revealed by genomics approaches. However, in this review we compare the mode of colonization of the two taxonomically distinct groups and show that their strategies have much in common.

Corresponding author: Francine Govers, Email: Francine.Govers(at)wur.nl

These abstracts were reprinted with the kind permission of the American Phytopathological Society (www.apsnet.org), the American Society for Microbiology (http://journals.asm.org), Blackwell Publishing (www.blackwellpublishing.com), and Elsevier Science (http://www.elsevier.com).