Phytophthora Infestans Sporangia Research Articles

Phytophthora infestans Sporangia Produced in Culture and on Tomato Leaflet Lesions Show Marked Differences in Indirect Germination Rates, Aggressiveness, and Global Transcription Profiles.

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4°C (to induce indirect germination) or at 21°C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with a RPKM (reads per kilobase of exon model per million mapped reads) >1 were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared with sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were overrepresented, with almost half of the 355 effectors with RPKM >1 being up- or downregulated. DEGs of both isolates include nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.

Potencial de hibridação entre acessos de tomateiro para pré-melhoramento quanto à resistência à requeima

Resumo: O objetivo deste trabalho foi avaliar genitores com potencial de hibridação para o pré-melhoramento do tomateiro (Solanum lycopersicum) quanto à resistência à requeima. Foram utilizados seis acessos de tomateiro (BGH-2102, BGH-2117, BGH-2127, BGH-2130, BGH-2332 e BGH-2343) como genitores resistentes e 15 híbridos F1 originários destes genitores. Utilizou-se o delineamento de blocos ao acaso, com três repetições. As plantas foram inoculadas com uma mistura de esporângios de Phytophthora infestans, agente etiológico da requeima, na concentração de 5x103 esporângios mL-1. A área abaixo da curva de progresso da doença foi utilizada para avaliar a resistência. Realizou-se a análise dialélica, tendo-se considerado o efeito de genótipos como fixo. Estimou-se a capacidade geral e específica de combinação dos acessos. O padrão de resistência dos genitores e da maioria dos F1 foi o mesmo que o das testemunhas resistentes. Foram observados: variabilidade genética aditiva entre os genitores, predominância de efeitos gênicos não aditivos e desvios de dominância bidirecional no controle do caráter. A frequência de alelos favoráveis e divergentes para resistência à requeima é maior nos acessos BGH-2117, BGH-2127 e BGH-2343.

Evaluation of the RPi-ber late blight resistance gene for tuber resistance in the field and laboratory

With 4 figures and 1 table Abstract The purpose of this research was to evaluate the efficacy in tubers of a late blight resistance gene, RPi-ber, originating from Solanum berthaultii. Experiments were conducted in the field and laboratory. Inoculation of tubers in field trials occurred via sporangia produced on infections in the foliage. Laboratory trials were conducted by inoculating whole tubers and tuber slices with sporangia of Phytophthora infestans. In all trials, those progeny with RPi-ber were essentially immune to strains of P. infestans that were incompatible with RPi-ber in the foliage. However, even when tested against strains of P. infestans that were compatible with RPi-ber in the foliage, there was still a resistance effect at the RPi-ber region.

Effects of Soil-Applied Late Blight Foliar Fungicides on Infection of Potato Tubers by Phytophthora infestans.

Potato tuber infection was assessed under greenhouse and outdoor conditions when late blight foliar fungicides were applied to soil 24 h prior to soil infestation with a suspension of zoospores and sporangia of Phytophthora infestans. Spore viability of P. infestans in soil treated with various fungicides was determined using buried healthy whole tubers and by assaying infested soil applied to freshly cut tuber disks. Protection of tubers and tuber disks from infection was more effective when soil was treated with mancozeb, metiram, and cyazofamid than with other fungicides. Whole tuber infections were significantly less in soils treated with mancozeb, metiram, fluazinam, and fenamidone than when treated with distilled water. Infection of buried tubers and tuber disks was prevented for 3 to 5 days following a single soil application of mancozeb or metiram under outdoor conditions. The tuber disk method was more sensitive in determining the efficacy of a fungicide in inhibiting infection and spore viability than using whole buried tubers. However, both methods of determining viability may determine different modes of action of some fungicides that inhibit infection since whole tubers were not infected when protected by some fungicides but tuber disks were infected.

Use of a green fluorescent protein marker for studying splash dispersal of sporangia of Phytophthora infestans

The purpose of this study was to develop a new technique to evaluate the number of spores incorporated in splash droplets by the use of an engineered fluorescent pathogen strain and image analysis hardware and software. The inoculum source consisted of a tomato leaflet infected with a Phytophthora infestans strain transformed with the gene encoding the green fluorescent protein (GFP). Splash droplets formed after impact of incident drops on sporulating lesions were collected on microscope slides located at different distances from the source. Each slide was examined using a fluorescent microscope to visualize the GFP expressing sporangia. Photographs were taken and assessed using image processing to count the sporangia incorporated in each droplet. Data analysis confirmed characteristics of splash dispersal shown using other methods. The use of fluorescent sporangia also facilitates the selective detection and counting of viable (living) sporangia, and is a tool that can be use in the study of splash dispersed diseases.

Response of solanaceous cultivated plants and weed species to inoculation with A1 or A2 mating type strains of Phytophthora infestans

Hypersensitive response symptoms of late blight were induced when sporangia of Phytophthora infestans were inoculated on discs of excised leaf tissues of black nightshade, eggplant, green pepper, and tobacco. No symptoms due to disease or hypersensitive response were observed in stem piece tissues of these plant species, suggesting that these four are nonhosts for late blight. The Al (US-1), the A2 (US-8), and the more recent Al mating type (US-ll) strains infected both leaf and stem tissues of climbing and hairy nightshade, potato, and tomato but only leaf tissues of petunia. This is the first report of the new strains of P. infestans causing late blight symptoms on climbing nightshade and petunia in Canada. Based on infection levels, infection rates, and spore production, the US-8 and US-ll genotypes were similar and both were more aggressive than the US-1 genotype. Disease severity and sporulation were greater on potato inoculated with US-1 than for the other host plants and were the least on petunia. All susceptible plants were infected at about the same level with US-8, except for petunia, which had a much lower level of infection. Spore production for US-8 was less on tomato and petunia than on the other plants. Infection levels and spore production by US-ll on tomato were as great as or greater than on potato. These results demonstrate that the new strains of P. infestans, US-8 and US-ll, are generally more aggressive than the previous predominating strain, US-1, and that climbing and hairy nightshade and petunia may harbour late blight, while black nightshade, eggplant, green pepper, and tobacco plants do not.

Suppressed germination and early death of Phytophthora infestans sporangia caused by pectin, inorganic phosphate, ion chelators and calcium-modulating treatments

In laboratory experiments, chemical treatments were applied to sporangia of Phytophthora infestans incubated at 12 °C, conducive to cytoplasmic cleavage and release of zoospores (indirect germination), and at 20 °C, conducive to germination by hyphal outgrowth (direct germination). Both types of germination were suppressed by applying increasing concentrations (1–5 mM) of CaCl2 or MgCl2, or by low concentrations of pectin, inorganic phosphate, chelators (EGTA, BAPTA), calcium channel-blockers (lanthanum, gadolinium, verapamil) or compounds that interfere with intracellular calcium-mediated processes (trifluoperazine, caffeine). The suppression by some treatments was partly overcome by adding Ca2+ or Mg2+ in the early stages of incubation, but Ca2+ was usually more effective than Mg2+, and suppression at 20 °C was more easily overcome than at 12 °C. Pectin (0.1%) or BAPTA (5 mM) caused rapid death of sporangia at both 12 ° and 20 °, whereas EGTA (5 mM) or Na2HPO4 (5 mM) caused rapid death only at 12 °. The findings indicate that germinability and viability of P. infestans sporangia are strongly affected by the external availability of Ca2+ or other divalent cations, especially during zoosporogenesis.

Evidence for release of the elicitor arachidonic acid and its metabolites from sporangia of Phytophthora infestans during infection of potato

Arachidonic acid (AA), an abundant fatty acid in lipids of Phytophthora spp., elicits a series of host defence responses in potato that typically occurs following infection with incompatible races of Phytophthora infestans. To better understand initial processing of this unusual fungal elicitor during infection, we examined AA metabolism in potato leaves following their inoculation with sporangia that had been radiolabelled with 14C-AA. Sporangia incorporated 14C-AA primarily into phospholipids, and di- and tri-glycerides. Microautoradiography of these sporangia revealed that radioactivity was distributed throughout fungal cytoplasm and cell walls, and was often associated with lipid bodies. In potato leaf tissues treated with pure 14C-AA, radioactivity was found in polar and neutral lipid fractions but not in galactolipids. Analyses of lipids extracted from potato leaves sampled at various times after inoculation with 14C-AA-labelled sporangia revealed a similar distribution of radioactivity among the various lipid classes. Inoculated leaves also were examined by micro-autoradiography. Radioactivity was observed in host cells in both compatible and incompatible interactions, but was present earlier (9 h after inoculation) in the incompatible than in the compatible interaction (12 h after inoculation). Radioactivity was distributed among host cells throughout sections including areas several cell layers distant from the site of inoculation and fungal infection structures. These results provide direct evidence that AA and/or its metabolites are released rapidly from spores of P. infestans and have limited systemic movement within inoculation sites during early phases of infection of potato leaves.

Competition between oxadixyl-sensitive and -resistant field isolates of Phytophthora infestans on fungicide-treated potato crops

Potato crops (cv. Alpha) were grown in 19 walk-in plastic tunnels (33 m 2) in fall 1986 and spring 1987. Beginning 4–5 weeks after planting, crops were treated, at 2-week intervals, with oxadixyl or oxadixyl-containing fungicidal formulations. One day before the first spray, crops were inoculated with a 9:1 mixture of oxadixyl-sensitive (S) and -resistant (R) sporangia of Phytophthora infestans. Late blight development and the proportion of R in the fungal population were estimated for 8 weeks after inoculation. Results showed that in the absence of fungicide treatment the proportion of the R isolate increased to 85% and then, towards the end of the epidemic, declined to 28%. In crops treated with SAN371F (oxadixyl), SAN518F (oxadixyl + mancozeb), Pulsan and Sandocur-M (oxadixyl + mancozeb + cymoxanil), the proportion of R reached a final level of 100, 97, 34 and 22%, respectively. In sprayed crops, blight development was positively correlated ( R 2 = 0·86) with the mean proportion of R in the fungal population. Tuber yield was inversely ( R 2 = −0·98) related to the area under the disease progress curve.

Formation of multinucleate zoospores inPhytophthora infestans (Mont.) De Bary (Oomycetes, Pythiaceae)

Zoospore formation in sporangia ofPhytophthora infestans, the causal agent of potatoes downy mildew disease, was influenced by reduction of oxygen tension, by application of a respiratory poison (KCN), and by an inhibitor of cytokinesis (cytochalasine B). As demonstrated by different methods of light microscopy, in all three cases the percentage of morphologically abnormal, multinucleate, multiflagellate cells increased. High concentrations of KCN caused a complete inhibition of zoospore formation. These effects are interpreted as disorders of the process of cytokinesis during zoosporogenesis.

Bioassay and other laboratory methods for testing formulations of potato blight fungicides

SUMMARYThree laboratory methods are described for comparing formulations of potato blight fungicides; all use the upper surfaces of leaflets detached from glasshouse‐grown King Edward plants, and artificial rain in some form.Rain‐fastness (tenacity) was quickly estimated by measuring advancing acute contact angles of distilled water (representing rain) on dry spray deposits. Tenacity itself was measured by 'rain‐washing' deposits with 0·65 in. of tap water in 30 min. (light 'rain') or up to 2·5 in. in 40 mm. (heavy 'rain'). Contact angles and tenacities of copper oxychloride deposits were usually positively correlated. In bioassay tests, deposits were washed with 2·5 in. of heavy 'rain', inoculated with sporangia of Phytophthora infestans (Mont.) de Bary, and later classed as infected or not infected; results were analysed by the probit method. With copper oxychloride and triphenyl tin (fentin) acetate, differences in effectiveness of 2·5 times or more were usually significant when fifty to sixty leaflets were used per formulation.