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.     

A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.     

Characterization and mapping of Rpi1, a late-blight resistance locus from diploid (1EBN) Mexican Solanum pinnatisectum

Solanum is a diverse genus with over 200 species occupying a range of habitats from the Southwestern United States to Central Chile. Germplasm evaluations have focused on species that can be crossed with S. tuberosum, while Mexican diploid (2n = 2x = 24) Solanum species with an Endosperm Balance Number (EBN) of 1 have received less attention because of poor crossability due to their ploidy and EBN. Recent changes in Phytophthora infestans populations have increased the need for new sources of genetic resistance to this fungus. We have characterized resistance to P. infestans in the Mexican 2x(1EBN) species S. pinnatisectum. An interspecific hybrid between resistant S. pinnatisectum and susceptible S. cardiophyllum plants was backcrossed to S. cardiophyllum to generate a family segregating for late-blight resistance. The diploid (1EBN) genetic map generated with 99 RFLP markers revealed extensive synteny with previously published potato maps. A single dominant late-blight resistance locus (Rpi1) from S. pinnatisectum was mapped to chromosome 7, a region previously not associated with late-blight resistance. Characterization of the P.infestans isolate used for disease evaluations revealed that it possessed the avirulence gene corresponding to the R9 resistance locus, indicating that Rpi1 could possibly correspond to R9.     

Rapid determination of Phytophthora infestans sporangia using a surface plasmon resonance immunosensor

Phytophthora infestans is the cause of late blight disease in potato and is an economically important pathogen worldwide. Early disease detection is important to implement disease control measures. In this study a surface plasmon resonance (SPR) immunosensor for detection of P. infestans sporangia is presented. The specificity of an existing mouse monoclonal antibody (phyt/G1470 mAb) against P. infestans was investigated in plate-trapped antigen ELISA and in subtractive inhibition ELISA. No or only limited cross-reactivity was observed against representatives having air-borne spores from Ascomycetes, Deuteromycetes as well as Basidiomycetes. phyt/G1470 mAb was incorporated in a subtractive inhibition SPR assay, consisting of a pre-incubation of mAb and sporangia, a centrifugation step to remove sporangia-bound phyt/G1470 mAb and quantification of remaining phyt/G1470 mAb by SPR. Good intra- and interday assay variability was observed and the assay had a detection limit of 2.2x10(6) sporangia/ml. Analysis time was 75 min, which is superior to existing P. infestans detection methods.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.     

Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans.

A bioassay using Phytophthora infestans was developed to determine whether inhibitory proteins are induced in pathogen-inoculated plants. Using this bioassay, AP24, a 24-kilodalton protein causing lysis of sporangia and growth inhibition of P. infestans, was purified from tobacco plants inoculated with tobacco mosaic virus. Analysis of the N-terminal amino acid sequence identified AP24 as the thaumatin-like protein osmotin II. The sequence was also similar to NP24, the salt-induced protein from tomato. Subsequently, we purified a protein from tomato plants inoculated with P. infestans that had inhibitory activities identical to those of the tobacco AP24. The N-terminal amino acid sequence of this protein was also similar to those of osmotin and NP24. In general, both the tobacco and tomato AP24 caused lysis of sporangia at concentrations greater than 40 nanomolar and severely inhibited hyphal growth at concentrations greater than 400 nanomolar. Because both proteins were induced by pathogen inoculation, we discussed the possible involvement of these proteins as a plant defense mechanism.     

Teratogenic effects in early chick embryos of solanine and glycoalkaloids from potatoes infected with late-blight, Phytophthora infestans.

Solanine or a preparation of mixed glycoalkaloids from potatoes naturally infected with the late-blight fungus, Phytophthora infestans, was injected into fertile chicken eggs between 0 and 26 h of incubation, before formation of the neural tube. The embryos were examined after a total of 72 h of incubation. Various abnormalities were found, the most conspicuous being absence of the tail or trunk below the wing bud (rumplessness). A statistically significant proportion of the abnormal embryos showed malformations that seemed to be related to this condition; these included fluid- or blood-filled vesicles in the lower trunk or tail region on one or both sides of the neural tube. Such abnormalities were not observed in control embryos.     

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.     

Infection Potential of Hairy Nightshade (Solanum sarrachoides) by Phytophthora infestans and Late Blight Implications of the Alternate Host

Infection of hairy nightshade ( Solanum sarrachoides Sendt.) by Phytophthora infestans has been reported. However, the epidemiological significance of hairy nightshade to potato late blight is not well known. Disease development and infection rates of P. infestans were quantified on hairy nightshade relative to tomato (cv. Brandywine) and potato (cv. Shepody) hosts to evaluate infection potential at 14, 18, 22 and 26°C and 72, 82, 87, and 92% relative humidity (RH). The susceptibility of hairy nightshade to inoculum levels, weed ontogeny, and sporangia production potential were also investigated. Late blight development varied among hairy nightshade, tomato and potato hosts. Pathogen infection rates ranged from 0.0325 to 0.4674 gompits/day (unit for quantifying infection rates), and were significantly (P < 0.05) greater on potato and tomato than on hairy nightshade. Late blight severity was variably affected by RH. Disease levels on hairy nightshade varied with inoculum load; and ranged from 9 to 26% and 26 to 37% at low (5 × 10³) and high (25 × 10³) sporangia concentrations, respectively. Late blight was recorded irrespective of hairy nightshade ontogeny, and was significantly greater on 8–10 than 4–6-week-old plants. These results indicate that pathogen, environmental and host factors affect late blight development on hairy nightshade.     

Somatic hybrids <Emphasis Type="Italic">Solanum nigrum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis>: morphological assessment and verification of hybridity

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone. From a total of 59 regenerants, 40 clones survived and have been maintained in vitro on hormone-free MS/2 medium. Thirty-two somatic hybrids were identified by their intermediate morphology (leaves of nigrum type and flowers of tuberosum type) and verified by flow cytometry and random amplified polymorphic DNA (RAPD) patterns. The RAPD analysis of nuclear DNA confirmed the hybrid nature of 29 clones. Flow cytometry revealed a wide range of ploidy in the generated hybrids, from nearly the tetra- to decaploid level. Most of the hybrid clones were stable in vitro, grew vigorously in soil, and set flowers and parthenocarpic berries. However, all of the flowering hybrids were male-sterile. Nine hybrid clones produced tuber-like structures in soil. The most vigorous flowering somatic hybrids were selected for assessment of the late-blight resistance.     

Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes.

We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns of P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.     

Divinyl ether fatty acid synthesis in late blight-diseased potato leaves

We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems.     

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.     

Structural analysis and activation by fungal infection of a gene encoding a pathogenesis-related protein in potato

The structure, genomic organization, and temporal pattern of activation of a gene encoding a pathogenesis-related protein (PR1) in potato (Solanum tuberosum) have been analyzed. The gene is rapidly activated in leaves from the potato cultivar Datura, containing the resistance gene R1, in both compatible and incompatible interactions with appropriate races of the late-blight fungus Phytophthora infestans. Activation is also observed in leaves treated with fungal elicitor. The gene occurs in multiple, very similar copies and encodes a polypeptide (Mr = 25,054; pI = 5.5) that is classified as a PR protein by several criteria. Small fragments with great sequence similarity to portions of the two exons were found closely linked to the expressed gene, which altogether represents a simple case of genome organization in potato. The coding sequence of the prp1 gene and the deduced amino acid sequence are strikingly similar to the corresponding sequences of a 26-kDa heat shock protein from soybean.     

Cell cycle regulator Cdc14 is expressed during sporulation but not hyphal growth in the fungus-like oomycete Phytophthora infestans

Cdc14 proteins are important regulators of mitosis and the cell cycle. These phosphatases have been studied previously only in yeasts and metazoans, which grow by fission or budding. Here we describe a homologue (piCdc14) from the oomycete Phytophthora infestans, a primitive eukaryote lacking a classical cell cycle. PiCdc14 complements a cdc14ts mutant of Saccharomyces cerevisiae and may function like other Cdc14 proteins, but displays a strikingly different pattern of expression. Whereas previously studied Cdc14 genes are constitutively transcribed, piCdc14 is not expressed during normal growth but instead only during asexual sporulation. In transformants of P. infestans expressing a fusion between the piCdc14 promoter and the -glucuronidase reporter, expression was first detected in sporangiophore initials, persisted in sporangiophores bearing immature sporangia, and later became restricted to mature sporangia. After germination, expression ended a few hours before the resumption of mitosis in hyphae emerged from the spores. Homology-dependent silencing experiments supported an essential role of piCdc14 in sporulation. It is proposed that the function of piCdc14 may be to synchronise nuclear behaviour during sporulation and maintain dormancy in spores until germination. These results help illuminate the process of sporulation in oomycetes and the evolution of the cell cycle in eukaryotes.