Suitability of Ten Plant Baits for the Rapid Detection of Pathogenic Pythium Species in Hydroponic Crops

Ten types of plant baits were tested in the laboratory to assess their capacity to detect pathogenic Pythium species. These were orange tree leaves, tomato leaves, pepper leaves, geranium leaves, Bermuda grass leaves, pine needles, immature carnation petals, hemp-seed cotyledons, pepper and cucumber fruits. The Pythium spp. tested were P. aphanidermatum, P. irregulare and Pythium group F from hydroponic market garden crops in the Poniente region of Almería (south-east Spain). The test consisted of observing the velocity at which five baits were colonized and the day of colonization of the first bait. Results indicated that the slowest baits to be infected were immature carnation petals and pine needles. These two, together with Bermuda grass leaves, were also the baits infected in lowest number, such that practically no further infection was produced in the baits after the fifth day of contact with the inoculated water. The other plant baits tested were equally suitable for detection of Pythium spp. over the first two days, although only orange leaves and hemp-seed cotyledons were infected on the first day.     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.     

Practical method combining loop-mediated isothermal amplification and bait trap to detect <Emphasis Type="Italic">Pythium helicoides</Emphasis> from hydroponic culture solutions

Two detection methods combining loop-mediated isothermal amplification (LAMP) and a bait trap were developed to detect Pythium helicoides in greenhouses containing roses, miniature roses, and poinsettias in hydroponic culture systems. In “Bait-LAMP”, a crude extract derived from perilla seeds as the bait was used in the LAMP reaction, whereas in the “Bait culture-LAMP”, a crude extract of mycelia grown out from perilla seeds onto Pythium-selective medium served as the bait. The two methods are simple and rapid for practical monitoring of P. helicoides in hydroponic culture systems.     

Pythium rot of chingensai ( Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of chingensai (Brassica campestris L. chinensis group) in Okayama Prefecture in 2000. The causal fungi were morphologically identified as Pythium ultimum Trow var. ultimum and P. aphanidermatum (Edson) Fitzpatrick. This is the first report of rot caused by Pythium species on chingensai. We named this disease Pythium rot of chingensai.     

Biological control of pythium root rot of chrysanthemum in small-scale hydroponic units

The capacity of several strains of root-colonizing bacteria to suppressPythium aphanidermatum, Pythium dissotocum and root rot was investigated in chrysanthemums grown in single-plant hydroponic units containing an aerated nutrient solution. The strains were applied in the nutrient solution at a final density of 10⁴ CFU ml⁻¹ and 14 days later the root systems were inoculated withPythium by immersion in suspensions of 10⁴ zoospores ml⁻¹ solution. Controls received no bacteria, noPythium, or one of thePythium spp. but no bacteria. Strain effectiveness was estimated based on percent roots colonized byPythium and area under disease progress curves (AUDPC). In plants treated respectively withPseudomonas (Ps.)chlororaphis 63-28 andBacillus cereus HY06 and inoculated withP. aphanidermatum, root colonization by the pathogen was 83% and 72% lower than in the pathogen control, and AUDPC values were reduced by 61% and 65%. ForP. dissotocum, the respective strains reduced root colonization by 87% and 91%, and AUDPC values by 70% and 90%. In plants treated respectively withPs. chlororaphis Tx-1 andComamonas acidovorans C-4-7-28, root colonization byP. aphanidermatum was 84% and 80% lower than in the controls and AUDPC values were reduced by 66% and 57%; these strains did not suppressP. dissotocum. Burkholderia gladioli C-2-74 andC. acidovorans OCR-7-8-38, respectively, suppressed colonization of roots byP. dissotocum by 74% and 86%, and reduced AUDPC values by 60% and 70%, but were ineffective againstP. aphanidermatum. C. acidovorans OCR-7-8-39 reduced colonization and AUDPC values ofP. aphanidermatum by 57% and 42%, respectively.Pseudomonas corrugata 13,Ps. fluorescens 15 and JZ12, and three additional strains ofC. acidovorans were weakly or nonsuppressive againstP. aphanidermatum. Strains that reduced AUDPC values forP. aphanidermatum orP. dissotocum when applied at 10⁴ CFU ml⁻¹ were 11%–39% less effective at 10³ CFU ml⁻¹. Four tested strains (Ps. chlororaphis 63-28,Ps. chlororaphis Tx-1,B. cereus HY06, andB. gladioli C-7-24) in most instances suppressed root colonization and lowered AUDPC values ofP. aphanidermatum when applied at 14, 7 or 0 days before inoculation, but reduction of the respective variables was generally greater when the strains were applied at 14 days (63%–87% and 75%–78%) or 7 days (44%–47% and 31%–88%) than at 0 days (14%–31% and 23%–62%) before inoculation.Ps. chlororaphis Tx-1,Ps. chlororaphis 63-28 andB. cereus HY06 significantly suppressedP. aphanidermatum whether the temperature of the nutrient solution was high (32°C) or moderate (24°C). Taken together, the observations suggest thatPs. chlororaphis 63-28,B. cereus HY06,Ps. chlororaphis Tx-1,B. gladioli C-2-74 andC. acidovorans OCR-7-8-38 have the potential for controlling Pythium root rot in hydroponic chrysanthemums. http://www.phytoparasitica.org posting Jan. 24, 2007.     

Root and stem rot of chrysanthemum caused by five <Emphasis Type="Italic">Pythium</Emphasis> species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.     

<Emphasis Type="Italic">Pythium</Emphasis> and <Emphasis Type="Italic">Phytophthora</Emphasis> species associated with root and stem rots of kalanchoe

Pythium and Phytophthora species were isolated from kalanchoe plants with root and stem rots. Phytophthora isolates were identified as Phytophthora nicotianae on the basis of morphological characteristics and restriction fragment length polymorphism (RFLP) analysis of the rDNA-internal transcribed spacer regions. Similarly, the Pythium isolates were identified as Pythium myriotylum and Pythium helicoides. In pathogenicity tests, isolates of the three species caused root and stem rots. Disease severity caused by the Pythium spp. and Ph. nicotianae was the greatest at 35°–40°C and 30°–40°C, respectively. Ph. nicotianae induced stem rot at two different relative humidities (60% and >95%) at 30°C. P. myriotylum and P. helicoides caused root and stem rots at high humidity (>95%), but only root rot at low humidity (60%).     

Pathogenicity ofPythium species on cucumber in peat-sand,rockwool and hydroponics

Pythium spp. that cause damping-off of seedlings also can cause root rot of older plants and lead to yield reductions. This can be especially severe in soilless cultures where the fungus can spread easily with the nutrient solution. 39Pythium isolates obtained from discolored roots were assayed for their ability to cause damping-off on cucumber seedlings in sand-peat and for their pathogenicity in soilless culture of cucumber in rockwool or hydroponic solution. Isolates ofPythium aphanidermatum, P. irregulare, P. sylvaticum andP. ultimum were highly pathogenic in sand-peat, but onlyP. aphanidermatum strains were pathogenic in soilless conditions and led to root decay, plant death in rockwool culture and growth reduction in hydroponic culture. One strain ofP. aphanidermatum significantly reduced the yield of cucumber grown in rockwool under conditions similar to those of commercial cultures.     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Monitoring by real-time PCR of three water-borne zoosporic Pythium species in potted flower and tomato greenhouses under hydroponic culture systems

The high-temperature-tolerant Pythium species P. aphanidermatum, P. helicoides, and P. myriotylum cause serious diseases in many crops under hydroponic culture systems in Japan. Control of the diseases is difficult because these zoosporic pathogens spread quickly. In this study, a real-time PCR method was developed for monitoring the spread of zoospores of the three pathogens. Specific primers and TaqMan probes were established using the internal transcribed spacer regions of the rDNA. Specificity was confirmed using known isolates of each species and closely related non-target species. The sensitivity of DNA detection was 10 f. for each pathogen. 10 f. DNA corresponded to 4 P. aphanidermatum, 3 P. myriotylum, and 4 P. helicoides zoospores, respectively. Therefore, this real-time PCR method was used to evaluate and monitor zoospores in the nutrient solutions of ebb-and-flow irrigation systems for potted flower production and closed hydroponic culture systems for tomato production. The results indicated that the pathogens were present in the hydroponic culture systems throughout the year, and spread before disease occurrence.     

Characterisation of fungal pathogens causing basal rot of lettuce in Belgian greenhouses

Basal rot is a common disease in lettuce greenhouses. A 3-year study on the diversity of pathogens associated with basal rot in Belgium was carried out. A total of 150 isolates were collected originating from 56 greenhouses. Four pathogens appeared to be involved. Rhizoctonia solani was found to be the causal agent at 23 locations, Sclerotinia spp. at 14, Botrytis cinerea at 17 and Pythium spp. at seven. The isolates of R. solani were further characterised to anastomosis groups and subgroups using morphological characteristics, pectic zymogram and PCR-RFLP. Five anastomosis groups could be distinguished: AG1-1B, AG4 HGI, AG10, AG2-1, AG2-1 Nt and AG3, with isolates of AG4 HGI and AG1-1B being the most prevalent and the most aggressive. Sclerotinia sclerotiorum was found at 13 locations, while S. minor was found at only one location. Based on ITS-sequencing Pythium isolates were assigned to three different species. At 20°C, isolates of all pathogens were able to cause lesions on detached lettuce leaves, except isolates of R. solani AG3 and AG2-1 Nt. A correlation could be found between the occurrence of the pathogens and the growing season. Botrytis cinerea was the most common pathogen in winter, whereas R. solani was most frequently isolated in summer. Sclerotinia spp. and Pythium spp. were isolated in spring, summer and autumn. The information obtained in this study will be most useful in the development of an alternative control strategy for causal agents of basal rot.     

三类植物卵菌的全基因组比较

为有效防治由不同类型卵菌引起的植物病害,该研究对数据库中的霜霉、疫霉和腐霉3类不同卵菌的全基因组数据进行序列特征分析、同源性分析和共线性分析以及同源差异基因富集通路分析。结果显示,霜霉、疫霉和腐霉来自独立的谱系,且疫霉与霜霉的亲缘关系较近;疫霉的致病相关基因数量最多,主要有RxLR、CRN、NPP、NF和PcF基因家族,霜霉的致病相关基因数量次之,腐霉的致病相关基因数量最少;3类卵菌共确定了13 392个同源基因,其中3类卵菌均共有的同源基因为3 786个,不同菌种的同源基因数量差异较大,整体表现为疫霉>霜霉>腐霉;同源差异基因富集程度最高的前20个通路主要是基础代谢的通路和中间代谢通路,其中抗坏血酸和藻酸盐代谢、ABC转运蛋白和果糖和甘露糖代谢相对富集程度较高,基因数目也较多。     

Evaluation of plant growth-promoting rhizobacteria for biological control of pythium root rot of cucumbers grown in rockwool and effects on yield

Three strains ofPseudomonas fluorescens (63-49, 63-28, and 15), one strain ofPseudomonas corrugata (13) and one strain ofSerratia plymuthica (R1GC4) were tested on rockwool-grown cucumbers for their ability to reduce Pythium root-rot caused byPythium aphanidermatum. These strains were previously selected for biocontrol ability from collections of >4000 bacteria. Strains 63-49 and 63-28 were tested on cucumber plants grown in rockwool in two replicatedPythium-inoculated trials conducted in British Columbia (B.C). Another inoculated, replicated trial was conducted in Quebec with all five strains. Cucumber yields (fruit number and weight) were measured over a ten-week harvest period. Strain 63-49 caused an early promotion of plant growth and increased cucumber yields at early harvests. No measurable effect ofPythium inoculation on disease development was observed in the Quebec trial, due to unfavourable cool weather. However, 63-49 significantly increased the total number of cucumbers (12%) and cucumber weight (18%), compared to the non-treated control. Strains 13, 15 and R1GC4 slightly increased the cumulative cucumber yields, but strain 63-28 had no effect. In the B.C. trial, inoculation withP. aphanidermatum reduced the number and weight of cucumbers by 27%. Treatments ofPythium-inoculated cucumbers with 63-49 significantly increased fruit number and weight by 18%, compared to thePythium-inoculated control. Strain 63-28 increased the cumulative number of cucumbers over time, compared to thePythium-inoculated control, but the increase was less than with 63-49. The use ofPseudomonas spp. in rockwool-grown cucumbers can increase yields, both in the presence and absence of Pythium root rot, and with variable seasonal conditions and disease pressures.     

Specific detection of<Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA

Pythium aphanidermatum causes damping-off and root rot of vegetable crops in hydroponic systems. A DNA probe was isolated and modified from a library ofHindIII-digested mitochondrial DNA ofP. aphanidermatum that strongly hybridized to DNA ofP. aphanidermatum and weakly hybridized to DNA ofPythium deliense. Cross-hybridizing sequences were absent from DNA of plants and other related fungi. The probe detected as little as 5 ng ofP. aphanidermatum DNA and 250 ng ofP. deliense DNA in slot-blot assays.P. aphanidermatum was detected by a hybridization assay of total DNA extracted directly from infected roots. A pair of oligonucleotide primers P1 and RP2, which allowed amplification of a specific 0.65 kb DNA fragment ofP. aphanidermatum using polymerase chain reaction (PCR), was designed from a specific DNA probe. Specific amplification of this fragment fromP. aphanidermatum was highly sensitive, detecting template DNA as low as 0.1 pg total DNA by booster PCR. Specific booster PCR amplification using P1 and RP2 was successful in detectingP. aphanidermatum in naturally infected nutrient solution and roots of vegetables in a field hydroponic system. http://www.phytoparasitica.org posting Sept. 22, 2002.     

An in-situ baiting bioassay for detecting Phytophthora species in irrigation runoff containment basins

Experiments were conducted in irrigation runoff containment basins to assess the effects of bait species ( Camellia japonica , Ilex crenata or Rhododendron catawbiense ), bait type (whole leaf vs. leaf disc), baiting duration (1, 2, 7 or 14 days), baiting depth and growth media (modified PARP-V8 or PARPH-V8) on the recovery of Phytophthora species. A two-rope, flexible bait-deployment system was compared with a one-rope fixed system for bait stability at designated locations and depths. A total of 907 Phytophthora isolates were subjected to PCR-based single-strand conformation polymorphism (PCR-SSCP) analysis to identify to species level. Seven distinct SSCP patterns representing six morphospecies: P. citricola (Cil I), P. citrophthora (Cip I), P. hydropathica (Hyd), P. insolita (Ins), P. megasperma (Meg I & II) and an unidentified Phytophthora species were identified. Irrespective of culture medium, 7 days of baiting with rhododendron leaves consistently resulted in the recovery of the greatest diversity and populations of Phytophthora species with minimum interference from Pythium species. The flexible bait-deployment system was superior to the fixed system, minimizing the risk of bait loss and dislocation of baiting units and allowing baits to remain at designated depths from the surface under inclement weather.     

Conduciveness of different soilless growing media to <Emphasis Type="Italic">Pythium</Emphasis> root and crown rot of cucumber under near-commercial conditions

Substrates made from rockwool, coir dust, pumice and perlite were compared for conduciveness to Pythium root and crown rot in cucumber under near-commercial conditions. Rockwool slabs of 7 cm height were more conducive to the Pythium disease than coir dust slabs, pumice or perlite under these conditions. Temperature, oxygen concentration and water content were determined in the substrates to explain differences in conduciveness between the inorganic substrates rockwool, pumice and perlite by differences in the physical conditions. Temperature and oxygen concentration could not explain the differences but the higher disease level on rockwool was associated with a much higher water content of this substrate as compared to coir dust, pumice and perlite. Increasing the height of the substrate from 7 to 14 cm greatly decreased the percentage of diseased plants due to the Pythium disease on rockwool but had no effect on the level of disease on perlite when the substrate had been infested 4 cm below the planting hole. This difference in response in substrate height between rockwool and perlite could be explained by a much larger decrease in water content with substrate height in the rockwool than in the perlite substrate. Temperature in the substrates were above 30 °C for more than 6 h on sunny days in June and reached maximum values of 35 °C or more. These temperatures are highly favourable for the pathogen P. aphanidermatum but will have adverse effects on most biocontrol strains.     

Adzuki bean leaf infection by <Emphasis Type="Italic">Phytophthora vignae</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> and resistance evaluation using detached leaves inoculated with zoospores

We examined the response of adzuki bean leaves to infection by Phytophthora vignae f. sp. adzukicola and determined whether inoculated leaves can be used to evaluate cultivar resistance. Detached adzuki bean leaves were inoculated with zoospores, and the resulting symptoms were diagnosed. Resistant reactions were characterized by dark brown, speckled lesions or a lack of symptoms, while susceptible reactions were characterized by water-soaked spreading lesions. In an inoculation experiment using a combination of three differential cultivars and three races, the response of 10-day-old primary leaves accurately differentiate between race-specific resistance and susceptibility of adzuki cultivars.     

Pathogenicity of <Emphasis Type="Italic">Stemphylium vesicarium</Emphasis> from different hosts causing brown spot in pear

Stemphylium vesicarium (teleomorph: Pleospora herbarum) is the causal agent of brown spot disease in pear. The species is also able to cause disease in asparagus, onion and other crops. Saprophytic growth of the fungus on plant debris is common. The objective of this study was to investigate whether isolates of S. vesicarium from different hosts can be pathogenic to pear. More than hundred isolates of Stemphylium spp. were obtained from infected pear fruits, dead pear leaves, dead grass leaves present in pear orchard lawns as well as from necrotic leaf parts of asparagus and onion. Only isolates originating from pear orchards, including isolates from dead grass leaves, were pathogenic on pear leaves or fruits in bioassays. Non-pathogenic isolates were also present in pear orchards. Stemphylium vesicarium from asparagus or onion, with one exception, were not pathogenic to pear. Analysis of the genetic variation between isolates using Amplified Fragment Length Polymorphism (AFLP) showed significant concordance with host plants. Isolates from asparagus or onion belonged to clusters separate from the cluster with isolates from pear or grass leaves collected in pear orchards. Multilocus sequencing of a subset of isolates showed that such isolates were similar to S. vesicarium.     

A Cucumber mosaic virus isolated from Momordica charantia L.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.     

Biochemical and histochemical studies on the digestive gland of Eobania vermiculata snails treated with carbamate pesticides

In this study the land snail (Eobania vermiculata), one of the most abundant gastropod of Egyptian farms, was investigated to determine the lethal toxic action of methomyl and methiocarb and to identify the biochemical and histochemical changes as a function of sublethal dose or concentration on the digestive gland of this snail under laboratory conditions, using topical application and baiting techniques. The results showed that methomyl exhibited greater efficacy than did methiocarb against the snails in both techniques. However, higher mortality rates were obtained in the topical application technique than in the poison bait experiments. Biochemical and histochemical examinations revealed that treatment of the snails with methomyl and methiocarb either by topical application or toxic baits caused significant decrease in carbohydrate, lipid and protein contents. This decrease was also more obvious after topical application than after baiting technique, and methomyl was found to be more toxic than methiocarb.