Dual-Transcriptomic, Microscopic, and Biocontrol Analyses of the Interaction Between the Bioeffector Pythium oligandrum and the Pythium Soft-Rot of Ginger Pathogen Pythium myriotylum

Biological control is a promising approach to suppress diseases caused by Pythium spp. such as Pythium soft rot of ginger caused by P. myriotylum. Unusually for a single genus, it also includes species that can antagonize Pythium plant pathogens, such as Pythium oligandrum. We investigated if a new isolate of P. oligandrum could antagonize P. myriotylum, what changes occurred in gene expression when P. oligandrum (antagonist) and P. myriotylum (host) interacted, and whether P. oligandrum could control soft-rot of ginger caused by P. myriotylum. An isolate of P. oligandrum, GAQ1, recovered from soil could antagonize P. myriotylum in a plate-based confrontation assay whereby P. myriotylum became non-viable. The loss of viability of P. myriotylum coupled with how P. oligandrum hyphae could coil around and penetrate the hyphae of P. myriotylum, indicated a predatory interaction. We investigated the transcriptional responses of P. myriotylum and P. oligandrum using dual-RNAseq at a stage in the confrontation where similar levels of total transcripts were measured from each species. As part of the transcriptional response of P. myriotylum to the presence of P. oligandrum, genes including a subset of putative Kazal-type protease inhibitors were strongly upregulated along with cellulases, elicitin-like proteins and genes involved in the repair of DNA double-strand breaks. In P. oligandrum, proteases, cellulases, and peroxidases featured prominently in the upregulated genes. The upregulation along with constitutive expression of P. oligandrum proteases appeared to be responded to by the upregulation of putative protease inhibitors from P. myriotylum, suggesting a P. myriotylum defensive strategy. Notwithstanding this P. myriotylum defensive strategy, P. oligandrum had a strong disease control effect on soft-rot of ginger caused by P. myriotylum. The newly isolated strain of P. oligandrum is a promising biocontrol agent for suppressing the soft-rot of ginger. The dual-RNAseq approach highlights responses of P. myriotylum that suggests features of a defensive strategy, and are perhaps another factor that may contribute to the variable success and durability of biological attempts to control diseases caused by Pythium spp.

Pythium myriotylum is recovered most frequently from Pythium soft-rot infected ginger rhizomes in China

Pythium soft rot is a major soil-borne disease of crops such as ginger (Zingiber officinale). Our objective was to identify which Pythium species were associated with Pythium soft-rot of ginger in China, where approximately 20% of global ginger production is from. Oomycetes infecting ginger rhizomes from seven provinces were investigated using two molecular markers, the internal transcribed spacer (ITS) and cytochrome c oxidase subunit II (CoxII). In total, 81 isolates were recovered and approximately 95% of the isolates were identified as Pythium myriotylum and the other isolates were identified as either P. aphanidermatum or P. graminicola. Notably, the P. myriotylum isolates from China did not contain the SNP in the CoxII sequence found previously in the P. myriotylum isolates infecting ginger in Australia. A subset of 36 of the isolates was analyzed repeatedly by temperature-dependent growth, severity of disease on ginger plants and aggressiveness of colonization of ginger rhizome sticks. In the pathogenicity assays, 32/36 of the isolates were able to significantly infect and cause severe disease symptoms on the ginger plants. A range of temperature-dependent growth, disease severity and aggressiveness in colonization was found with a significant moderate positive correlation between growth and aggressiveness of colonization of the ginger sticks. This study identified P. myriotylum as the major oomycete pathogen in China from infected ginger rhizomes and suggests that P. myriotylum should be a key target to control soft rot of ginger disease.

Pythium Soft Rot Management in Ginger (Zingiber officinale Roscoe) – A Review

Ginger crop is affected by various diseases. Among them rhizome/soft rot is the most damaging one and main production constraint in ginger growing areas. This disease is mainly caused by the Pythium spp. along with association of some others micro-organisms. The severity of Pythium soft rot disease is influenced by different factors related to seed, environment and soil. This study was focused on Pythium soft rot of ginger with special reference to different management strategies. Different cultural measures viz. seed rhizome treatment before storage and sowing, selection of disease free seed rhizome, sowing time, application of soil amendments, good drainage of soil, soil solarization etc. are the important measures for management of ginger soft rot. Seed treatment and soil drenching are the two options of chemical control of soft rot. Seed rhizomes treated with fungicides azoxystrobin 25%, tebuconazole 25.9%, copper oxychloride 50%, carbendazim 50%, propiconazole 25%, metalaxyl-M 4% + mancozeb 64%, metiram 55% + pyraclostrobin 5%, carbendazim 12% + mancozeb 63%, tebuconazole 25% + trifloxystrobin 25% and metalaxyl 8% + mancozeb 64% resulted in effective management. Different fungicide formulations viz. carbendazim 50%, copper oxychloride 50%, metalaxyl-M 4% + mancozeb 64%, metiram 55% + pyraclostrobin 5%, carbendazim 12% + mancozeb 63%, tebuconazole 25% + trifloxystrobin 25% etc. found effective for spraying. Seed treatment and application of Trichoderma spp. found suitable for effective biological management.

First Report in China of Soft Rot of Ginger Caused by Pythium aphanidermatum

Ginger (Zingiber officinale Roscoe) is an important commercial crop planted on more than 13,000 ha annually in Anqiu city, Shandong Province, China. From 2010 to 2011, the incidence of Pythium soft rot disease on cv. Laiwu Big Ginger reached 40 to 75% in Anqiu and yield losses of up to 60% were observed. The disease symptoms included brown spots on ginger rhizomes followed by soft rot, stems and leaves above ground becoming withered and yellow, and water soaking on the collar region. The soft rot did not produce offensive odors, which is different from bacterial rots (2). Forty symptomatic rhizomes were sampled from eight farms. Martin's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water for 30 s and plated on Martin's selective medium at 26°C in a chamber without light. Colonies grew with cottony aerial mycelium. Main hyphae were 5.7 to 9.6 μm wide. Globose sporangia consisting of terminal complexes of swollen hyphal branches were 11.4 to 18.3 μm wide. The average diameter of zoospores was 9.2 μm. The oogonia were globose and smooth, with a diameter of 21 to 33 μm. The sequences of the rRNA gene internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of five isolates were amplified using primers ITS1 and ITS4 (4), and the nucleotide sequence was the same as isolate No. 2, which was deposited in GenBank (Accession No. KC594034). A BLAST search showed 99% identity with Pythium aphanidermatum strain 11-R-8 (Accession No. JQ898455.1). Pathogenicity tests of five isolates were carried out in a greenhouse. Sixty plants (cv. Laiwu Big Ginger) were grown for 30 days in plastic pots (diameter 20 cm) in sandy soil (pH 5.48) and inoculated. Ten plants were used as untreated controls. Five isolates were grown on Martin's liquid medium for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of the five isolates were adjusted with deionized water to 1 × 108 CFU/ml and injected with a syringe into the soil around the rhizome of the plants. Plants were then placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. The inoculated isolates were recovered from the diseased rhizomes, confirming their pathogenicity. To our knowledge, this is the first report of ginger Pythium soft rot caused by P. aphanidermatum in China. Ginger Pythium soft rot caused by P. myriotylum is reported in Taiwan (3). References: (1) F. N. Martin. Page 39 in: The Genus Pythium. American Phytopathological Society, St. Paul, MN, 1992. (2) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December 1964. (3) P. H. Wang. Lett. Appl. Microbiol. 36:116, 2003. (4) T. J. White. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Assessment of Chemical Induction of Acquired Resistance to Pythium Fruit Rot in Field-grown Cucumber

Systemic acquired resistance (SAR) is a physiological defense response in plants conferring broad spectrum resistance to pathogens. SAR is inducible through infection by necrotizing pathogens or chemical inducers and involves the systemic activation of defense related genes. The objectives of this study were to evaluate resistance expression to Pythium soft rot in fruit of cucumber in response to foliar applications of 2,6-dichloro isonicotinic acid (INA), benzo(1,2,3)thiadiazole-7-carbothioc acid S-methyl ester (CGA 245704), or beta-amino-butyric acid (bABA). Excised leaves from three cucumber cultivars, Vlaspik, Sumpter and SMR 58, exhibited a resistance response to foliar applications of INA (50 ppm) and CGA 245704 (25 ppm) when challenge inoculated with Colletotrichum lagenarium. However, the reduction in lesion incidence by INA was 99% in Vlaspik and only 11% and 18% in Sumpter and SMR 58, respectively. bABA did not enhance resistance in vegetative tissues. Multiple foliar applications of INA and CGA 245704 prior to fruit set were found to be more effective than a single application. Harvested cucumber fruit, 3.0 to 5.0 cm in diameter, were wounded and challenge inoculated with Pythium sp.; no statistically significant differences in infection were observed between controls and fruit from chemically treated plots. In Vlaspik, however, INA and BTH did reduce the rot infection rate in fruit when foliar applications were made early in plant ontogeny, at the four-leaf stage. In addition, larger more mature fruit (4-5 cm diameter) exhibited slower lesion growth than less developed fruit (>3 cm). It is unclear if these differences were attributable to changes in physiological or anatomical factors. These findings indicate that the level of physiological resistance achievable through chemical induction in cucumber is a function of the specific pathogen and the plant organ being infected, i.e., leaves or fruit.

Currutaca: A Pythium Soft Rot of Xanthosoma and Colocasia spp. in Puerto Rico

The fungus Pythium ultimum Trow, was found the causal agent of a soft rot disease of taniers [Xanthosoma sagittaefolium (L.) Schott.] and taros [Colocasia esculenta (L.) Schott.]. A yellow pigmented bacterium occasionally associated with the rot turned out to be a saprophyte. Roots of diseased plants became mucilaginous and decayed rapidly. The infected rhizomes were soft and cheesy. The base of infected petioles were water-soaked and the leaves collapsed. The fungus was isolated in pure culture and grew well in most culture agar media, producing abundant sporangia at 20-30° C., but failed to grow at temperatures below 12° C. or above 36° C. Varieties of taniers and taro inoculated with P. ultimum showed differential degrees of susceptibility to infection when kept in an environment of 90-percent relative humidity and a temperature of 20° C. Definitive measures to control the disease effectively are not known. Use of clean "seed", planting in well drained soils, and crop rotation practices, however, have been recommended.