Pythium Torulosum Research Articles

Identification of Quantitative Disease Resistance Loci Toward Four Pythium Species in Soybean.

In this study, four recombinant inbred line (RIL) soybean populations were screened for their response to infection by Pythium sylvaticum, Pythium irregulare, Pythium oopapillum, and Pythium torulosum. The parents, PI 424237A, PI 424237B, PI 408097, and PI 408029, had higher levels of resistance to these species in a preliminary screening and were crossed with “Williams,” a susceptible cultivar. A modified seed rot assay was used to evaluate RIL populations for their response to specific Pythium species selected for a particular population based on preliminary screenings. Over 2500 single-nucleotide polymorphism (SNP) markers were used to construct chromosomal maps to identify regions associated with resistance to Pythium species. Several minor and large effect quantitative disease resistance loci (QDRL) were identified including one large effect QDRL on chromosome 8 in the population of PI 408097 × Williams. It was identified by two different disease reaction traits in P. sylvaticum, P. irregulare, and P. torulosum. Another large effect QDRL was identified on chromosome 6 in the population of PI 408029 × Williams, and conferred resistance to P. sylvaticum and P. irregulare. These large effect QDRL will contribute toward the development of improved soybean cultivars with higher levels of resistance to these common soil-borne pathogens.

First Report of Pythium torulosum Causing Corn Root Rot in Northeastern China.

In October 2017, we collected five soil samples from each of several fields with a history of severe corn (Zea mays) seedling disease in Heilongjiang province of China. Affected seedlings were wilted with severe root rot, and a high incidence of seedling death was observed in the fields. Corn seeds were seeded in the collected soil samples and grown in a growth chamber for 21 days set at the following incubation temperatures: 21℃/7℃ for 6 days, 10℃/3℃ for 4 days, 16℃/7℃ for 5 days, 20℃/20℃ for 6 days (16 h/8 h, light/dark) (Tang et al. 2019). The corn seedlings in the growth chamber showed the same symptoms observed in the field as mentioned above. Corn root rot samples were collected from several symptomatic plants in the growth chamber to isolate the possible pathogen. Symptomatic roots were washed in 0.5% NaOCl for 2 min, rinsed in sterile water and cut into 1-2 mm segments and then plated on corn meal agar amended with pimaricin (5 μg/ml), ampicillin (250 μg/ml), rifampicin (10 μg/ml), pentachloronitrobenzene (50 μg/ml), and benomyl (10 μg/ml) (PARP+B), which is selective for oomycetes (Jeffers and Martin 1986). After 3 days of incubation in the dark at 25℃, colonies were transferred to 10% V8 juice agar and incubated at 25℃ for 2 weeks. Six isolates were identified as Pythium torulosum based on the morphology of sexual and asexual structures following van der Plaats-Niterink's key (van der Plaats-Niterink 1981). On 10% V8 juice agar, the hypha were aseptate and colonies had filamentous sporangia with a dendroid or globose structure. The oogonia were globose or subglobose, laevis, terminal, rarely intercalary, ranging from 12-19 (average 16) μm. Antheridia were mostly sessile or brachypodous, and each oogonium was supplied by 1-2 antheridia cells. Oospores were globose, plerotic, ranging from 9-16 (average 13) μm. For the molecular identification, two molecular targets, the internal transcribed spacer (ITS) region of ribosomal DNA and cytochrome c oxidase subunit II (CoII), were amplified and sequenced using universal primer sets DC6/ITS4 (Cooke et al. 2000) and FM58/FM66 (Villa et al. 2006), respectively for one isolate, "copt". BLAST analyses of a 971 bp ITS segment amplified from copt (GenBank Accession No. MT830918) showed 99.79% identity with a P. torulosum isolate (GenBank Accession No. AY598624.2). For the COⅡ gene of copt, BLAST analyses of a 553 bp segment (GenBank Accession MT843570) showed 98.37% identity with P. torulosum isolate (GenBank Accession No. AB095065.1). Thus, the isolate, copt, was identified as P. torulosum based on morphological characteristics and molecular analysis. To confirm pathogenicity and Koch's postulates, a pathogenicity test was conducted as described by Zhang et al. (2000). Briefly, a 5 mm culture plug from the P. torulosum isolate, copt, was transferred to a 9-cm petri dish containing 20mL 10% V8 juice agar and incubated in the dark at 25℃ for 7 days. The culture was cut into small pieces and mixed with a sterilized soil mix (40% organic peat substrate, 40% perlite, and 20% soil) at a ratio of one petri dish per 100 g soil mix. Ten corn seeds were planted at a depth of 2 cm in a 500-mL pot containing the inoculated soil mix. The control pots were mock inoculated with plain 10% V8 juice agar. Pots were incubated in a greenhouse at temperatures ranging from 21 to 23℃. There were four replications. After 14 days, corn roots brown and rotted were observed, which was similar to those observed in the field and growth chamber. Control plants remained symptomless and healthy. P. torulosum copt was consistently re-isolated from the symptomatic roots. To our knowledge, this is the first report of P. torulosum causing root rot of corn in Northeastern China. Corn is an important crop in Heilongjiang and the occurrence of root rot caused by this pathogen may be a new threat to corn plants. There is a need to develop management measures to control the disease.

Resistance of the SoyNAM Parents to Seed and Root Rot Caused by Four Pythium Species

Some Pythium spp. cause damping off and root rot in soybeans and other crop species. One of the most effective management tools to reduce disease is host resistance; however, little is known about resistance in soybean to Pythium spp. The soybean nested associated mapping (SoyNAM) parent lines are a set of germplasms that were crossed to a single hub parent to create recombinant inbred line populations for the purpose of mapping agronomic traits. The SoyNAM parents were screened for resistance to Pythium lutarium, Pythium oopapillum, Pythium sylvaticum, and Pythium torulosum in separate assays to evaluate seed and root rot severity. Of the 40 SoyNAM parents, only 'Maverick' was resistant to the four species tested; however, 13 were resistant to three species. Other lines were resistant to two, one, or none of the species tested. Correlations between seed and root rot severity for the lines assessed were weak or insignificant. Results indicate that mechanisms of resistance to seed and root rot caused by Pythium spp. may not necessarily be the same.

Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR

ABSTRACT: We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates.

Attraction and oviposition responses of the fungus gnat Bradysia impatiens to microbes and microbe‐inoculated seedlings in laboratory bioassays

AbstractLaboratory tests were conducted to examine preferences of Bradysia impatiens Johannsen (Diptera: Sciaridae) larvae and adults for various microbes associated with greenhouse crops. Fungus gnat larvae and adults exhibited a preference for cultures of Pythium spp. over the medium used to grow the pathogens. Larvae also exhibited a preference for geranium seedlings infected with pathogenic Pythium spp. [P. aphanidermatum (Edson) Fitz., P. ultimum Trow, and P. irregulare Buis. (Oomycota: Peronosporales)] over non‐inoculated plants. Adult fungus gnats exhibited a strong ovipositional preference for the aforementioned Pythium spp. as well as a variety of other microorganisms, including the pathogenic fungus Thielaviopsis basicola (Berk. & Br.) (Ascomycota: Microascales), the geranium‐infecting bacterium Xanthomonas campestris pv. pelargonii (Brown) Dye (Proteobacteria: Xanthomonadales), the non‐pathogenic species Pythium torulosum Coker & P. Patt. and Pythium graminicola Subramaniam, the pathogen‐suppressive fungus Trichoderma harzianum Rifai (Ascomycota: Hypocreales), and the insect pathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales). Our study is the first to demonstrate that fungus gnats are attracted to and/or stimulated to oviposit by a wide array of living microorganisms both in pure culture and in association with plant seedlings. These findings have important implications with respect to the potential role of fungus gnats in plant pathogen transmission.

Development of Multiplex PCR to Detect FivePythiumSpecies Related to Turfgrass Diseases

The objective of this study was to develop multiplex PCR detection method for five Pythium species associated with turfgrass diseases, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium torulosum and Pythium vanterpoolii. Species-specific primers and two common primers were designed based on the sequences of the internal transcribed spacer region of ribosomal DNA. Another primer set by which all organisms would be amplified in 18S rDNA was used as a positive control. When these total nine primers were applied to the multiplex PCR, all species were individually discriminated in the mixture of five species culture DNA. Furthermore, all five Pythium species were detected in naturally infected plants using the multiplex PCR.

Distribution and Expression of Elicitin-like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin-like proteins (POD-1, POD-2, POS-1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D-type isolate containing POD-1 and POD-2, and the S-type isolate containing POS-1. We identified the genes encoding these elicitin-like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D-type isolate MMR2 was constructed and genomic regions containing the elicitin-like protein genes were identified. Southern blot analyses with probes derived from pod-1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D-type isolates carried pod-1, pod-2 and two oligandrin genes, termed oli-d1 and oli-d2, while the S-type isolates carried pos-1 and one oligandrin gene termed oli-s1. Phylogenetic analysis of POD-1, POD-2, POS-1, Oli-D1, Oli-D2 and Oli-S1 with the previously defined elicitins and elicitin-like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT-PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D-type and the S-type isolates, physical maps of the flanking regions around pod-1, pod-2, pos-1 and the oligandrin genes were constructed. The maps suggest that the D-type isolates may be derived from the S-type isolates due to gene duplication and deletion events.

Pathogenicity of Pythium Species Associated with Pythium Root Dysfunction of Creeping Bentgrass and Their Impact on Root Growth and Survival.

Symptoms resembling Pythium root dysfunction have been observed on golf course putting greens established with creeping bentgrass across the southeastern United States since 2002. Root isolations from 14 golf courses yielded 59 isolates of Pythium volutum and 16 isolates of Pythium torulosum. Pathogenicity of five isolates of P. volutum, two isolates of P. torulosum, and a combination of the two species was determined by inoculating mature 'A-1' creeping bentgrass plants. Inoculated plants were incubated for 4 weeks at 24/16°C (day/night) to permit root infection, then temperatures were increased to 32/26°C to induce foliar symptoms. No isolates impacted root depth, root mass, or foliar disease severity after 4 weeks at 24/16°C. After increasing the temperature to 32/26°C, isolates of P. volutum induced foliar disease severity ranging from 60 to 84%, whereas isolates of P. torulosum induced only 14 to 35% disease. Isolates of P. volutum consistently reduced root mass and root depth after 4 weeks at 32/26°C, but P. torulosum exhibited no effect. These results demonstrate that P. volutum is a pathogen of mature creeping bentgrass plants. Infections that occur during cool weather reduce the growth and survival of creeping bentgrass roots during hot weather and give rise to foliar symptoms.

First Report of Damping-Off of Wild Rice in California Caused by Pythium torulosum.

During 1994, damping-off of wild rice (Zizania palustris L.) in a single field in eastern Shasta County, CA resulted in near total stand failure. Since then, the disease was observed in at least 11 other fields with varying levels of stand loss. In all cases, the affected wild rice was grown as a volunteer crop following one or more years of wild rice production. Symptoms included a dark red discoloration and necrosis of the primary root followed by seedling death. When the red discoloration was limited to secondary roots, the plants often survived. Pythium torulosum, readily recovered from symptomatic roots by isolation on PARP media, was identified by morphological structures produced on grass blades in water (homothallic with smooth-walled oogonia, plerotic oospores, monoclinous antheridia, and inflated filamentous sporangia) and a 99.2% internal transcribed spacer sequence similarity of the rDNA (1). To complete Koch's postulates, inoculum of two isolates of P. torulosum grown on moistened cornmeal/sand (2%/98% [v/v]) for 3 weeks at 25°C were combined and mixed into sterilized sandy loam soil at a rate of 30 cm3 inoculum per liter of soil. Sterilized noninfested soil was used as a control treatment. Twenty wild rice seeds (cv. Franklin) were sown in each of four replicate 20-cm-diameter pots in each treatment. Plants were submerged in water and maintained in a greenhouse at 18 to 25°C. After 8 weeks, plants stands were reduced 50% in infested pots; dry weights of infected plants were reduced by 45% relative to the controls. The fungus was reisolated from symptomatic plants but not from the plants grown in noninfested soil. The experiment was repeated with similar results. To our knowledge, this is the first report of damping-off of wild rice caused by P. torulosum. Reference: (1) C. A. Levesque and W. A. M. DeCock. Mycol. Res. 108:1363, 2004.

Compatibility of systemic acquired resistance and microbial biocontrol for suppression of plant disease in a laboratory assay

AbstractSystemic acquired resistance (SAR) and microbial biocontrol each hold promise as alternatives to pesticides for control of plant diseases. SAR and Bacillus cereus UW85, a microbial biocontrol agent, separately suppress seedling damping‐off diseases caused by oomycete pathogens. The purposes of this study were to investigate how expression of SAR affected the efficacy of biocontrol by UW85 and if UW85 treatment of plants induced SAR. We devised a laboratory assay in which seedling damping‐off disease, induction of SAR, and growth of UW85 could be quantified. Seedlings of Nicotiana tabacum Xanthi nc were germinated on moist filter paper and transferred after 7 days to water agar plates (40 seedlings per plate). Zoospores of oomycete pathogens (Pythium torulosum, Pythium aphanidermatum, or Phytophthora parasitica) were applied at concentrations that caused 80% seedling mortality within 10 days. Seedling mortality was dependent on zoospore inoculum concentration. The level of disease suppression caused by treatment with UW85 depended on the UW85 dose applied. SAR was induced with 0.5‐mM salicylic acid or 0.1‐mM 2,6‐dichloroisonicotinic acid. Expression of an SAR‐related gene was confirmed by northern analysis with a probe prepared from a tobacco PR‐1a cDNA. Induction of SAR suppressed disease caused by each of the oomycete pathogens, but did not alter the growth of UW85 on roots. Treatment of seedlings with UW85 did not induce the expression of PR‐1a. The combination of induction of SAR and treatment with UW85 resulted in additive suppression of disease as measured by seedling survival.

Pathogenicity of Pythium torulosum to roots of Agrostis palustris in black-layered sand produced by the interaction of the cyanobacteria species Lyngbya, Phormidium, and Nostoc with Desulfovibrio desulfuricans

Studies were initiated to determine the pathogenicity of Pythium torulosum to Agrostis palustris roots growing in sand with subsurface black layer produced by the interaction of cyanobacteria and the sulfate-reducing bacterium Desulfovibrio desulfuricans. The interaction of P. torulosum with cyanobacteria and D. desulfuricans was also evaluated. Pythium torulosum decreased the dry weight of roots and shoots of A. palustris to 41 and 35%, respectively, of the control plants in the absence of black layer and the organisms responsible for its formation. The combination of P. torulosum and D. desulfuricans, in the absence of black layer, induced the most severe decrease in root (20% of controls) and shoot (25% of controls) dry weights. Damage to roots induced by P. torulosum in combination with various isolates of cyanobacteria, in the absence of black layer, was equal to that of P. torulosum alone; shoot dry weight loss was less than that caused by P. torulosum alone. Pathogenicity of P. torulosum to roots when combined with cyanobacteria and D. desulfuricans in the presence of black layer was the same as that with P. torulosum alone and in combination with cyanobacteria; shoot dry weight did not differ from that of P. torulosum combined with cyanobacteria. The presence of cyanobacteria with P. torulosum and D. desulfuricans in black-layered sand decreased root and shoot dry weight loss induced by the two latter organisms. Key words: anaerobic, black plug layer, golf greens, sulfate reduction.

Comparison of Physical Maps of Ribosomal DNA Repeating Units in Pythium, Phytophthora and Apodachlya

SUMMARY: A satellite band in CsCl-bisbenzimide density gradients of Pythium diclinum DNA was found to consist of ribosomal DNA (rDNA) repeating units with a complexity of 10.3 kilobases (kb). Similar satellite bands were also obtained from a morphologically diverse selection of species from the Pythiaceae: Pythium torulosum (10.4 kb), Pythium irregulare (11.8 kb), Pythium anandrum (10.6 kb), Pythium paddicum (10.6 kb) and Phytophthora cryptogea (11.2 kb). Physical maps were constructed using seven endonuclease restriction enzymes and the maps were aligned on the basis of nine conserved restriction sites in a 6 kb region which was shown to be homologous to a DNA plasmid probe containing ribosomal genes from Neurospora crassa. A map of the rDNA region in Apodachlya pyrifera (10.1 kb), a species external to the Pythiaceae (Leptomitales), was also constructed to serve as a taxonomic reference. Percentage sequence divergence values indicated that P. diclinum and P. torulosum are closely related, but not identical. P. anandrum and P. irregulare also appeared closely related in spite of morphological differences. P. paddicum and Phytophthora cryptogea were the most outlying of the six species of Pythiaceae, but all six species formed a tight cluster when considered against A. pyrifera.

An inverted repeat comprises more than three-quarters of the mitochondrial genome in two species of Pythiumm

Physical maps of mitochondrial DNA from Pythium torulosum (73 kb) and Pythium diclinum (70 kb) reveal that an exceptionally large proportion (75 and 80%, respectively) of the mitochondrial genomes of these oomycetes exists in the form of an inverted repeat. The size of the inverted repeat is more highly conserved between the two species than is the size of the small single-copy region. This indicates that the presence and size of an inverted repeat may be useful in oomycete systematics at the species level.