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.     

Bait method to detect Pythium species that grow at high temperatures in hydroponic solutions

Pythium helicoides, P. aphanidermatum and P. myriotylum are important pathogens that cause root rot of several crops in hydroponic culture and in ebb-and-flow irrigation systems. These species belong to a group of Pythium species that can grow at temperatures higher than 40°C. We developed a method for baiting these high-temperature Pythium species and evaluated its practicality to monitor their presence in nutrient solutions. Seeds of cucumber, tomato, radish, hemp, perilla and millet and leaves of bent grass and rose were tested as baits in hydroponic systems. Hemp, perilla and radish seeds and bent grass and rose leaves were more effective than the other baits for Pythium zoospores, and bent grass leaves were the most effective. In a sensitivity test, bent grass leaf traps (BLTs) detected three Pythium species after only a 1 day exposure to suspensions of 40 zoospores per liter of water, and the frequency of detection increased with zoospore density and with baiting period. A temperature of 38°C was optimum for the selective reisolation of the high-temperature Pythium species from the BLTs. The BLT was also tested with inoculated and noninoculated miniature roses that shared a recirculating nutrient solution. The pathogen was detected in the nutrient solution 23 days before the disease spread to the noninoculated roses. In addition, P. helicoides was detected 30 days before the disease was evident in a commercial greenhouse. The baiting method described here will be useful for monitoring high-temperature Pythium species in recirculating hydroponic culture systems.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).     

Wilt and root rot of poinsettia caused by three high-temperature-tolerant Pythium species in ebb-and-flow irrigation systems

Poinsettia plants growing in ebb-and-flow irrigation systems developed wilting and root rot during the summer growing seasons of 2010 in Gifu Prefecture and 2011 in Aichi Prefecture. Pythium species were isolated from roots with rot symptoms. The isolates were identified as P. helicoides and P. myriotylum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, these isolates caused severe wilting and root rot. This is the first report of poinsettia root rot disease caused by P. helicoides and P. myriotylum, although P. aphanidermatum was reported as a pathogen of poinsettia root rot. To better understand these diseases, we performed an epidemiological study of three high-temperature-tolerant Pythium species, P. aphanidermatum, P. helicoides and P. myriotylum. Disease incidence as a percentage of diseased plants was greatest at 35 °C for all three species. Disease severity using the rating scale of root rot was also highest at 35 °C, particularly with high zoospore inoculum densities (100.0 zoospores/mL). Although the disease incidence and severity were reduced at lower temperatures, the three Pythium species were able to cause disease at temperatures as low as 20 °C.     

Pythium rot of figmarigold (<Emphasis Type="Italic">Lampranthus spectabile</Emphasis>) caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

A new leaf rot disease was found on the leaves of figmarigold (Lampranthus spectabile). The causal organism, identified as Pythium aphanidermatum was found to cause the same symptoms after artificial inoculation and was then reisolated from the inoculated plants. We propose to name the disease Pythium rot of figmarigold.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Molecular genetic variability of Italian binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. isolates from strawberry

Fifty-eight binucleate Rhizoctonia isolates were collected over six years from strawberry plants displaying symptoms of black root rot in Italy. Almost all isolates were able to produce necrosis on strawberry roots, most of them also showed this ability on faba bean and, with lower frequency, on a crucifer and a cereal crop used in rotation with strawberry in Italy. The sequence alignment of Internal Transcribed Spacer (ITS) regions of 51 binucleate Rhizoctonia were analyzed and compared with a set of eight sequences representative of Rhizoctonia isolate Anastomosis Groups (AG) already found to be pathogenic on strawberry (AG-A, AG-G, AG-I and AG-F). The neighbour-joining tree, based on ITS region sequences, divided Italian strawberry Rhizoctonia isolates into two main clusters corresponding to AG-A and AG-G. The results were confirmed by hyphal anastomosis tests. The clustering obtained with the phylogenetic tree was also confirmed using PCR-Restriction Fragment Length Polymorphism of 28S rDNA to compare some isolates, defined as AG-A and AG-G on the basis of ITS region sequence analysis, with representative AG isolates pathogenic on strawberry. The AG-A and AG-G Rhizoctonia spp. were widespread in Italian strawberry-growing areas, although with different relative frequencies: AG-G was most frequent in northern (latitude 44°N) and AG-A in southern (latitude 39–40°N) Italy. Analysis of MOlecular VAriance, based on geographic location, showed that Rhizoctonia molecular variations between northern and southern Italy accounted for 36.6% of the total, but most of the variations (61%) occurred within each of the four geographical regions from where the isolates originated.     

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 of <Emphasis Type="Italic">Phytophthora</Emphasis> species on fruit trees in Bulgaria

Disease on fruit trees in Bulgaria caused by Phytopthora cactorum and P. citrophthora was found in the period 1998–1999. Leaves of some trees become reddish during July, and later in the season fall off. Infected trees die during the same season, or the next season. Observations on symptom development and spread of Phytophthora root and crown rot of fruit trees was undertaken from 1999 to 2009. Disease incidence is between 2% and 14% in some gardens and nurseries. The disease was registered in the regions of Plovdiv, Kjustendil, Sliven, Yambol, Karnobat, Bourgas and Svishtov. Samples from infected plant tissues were taken and isolations were done on selective PARP media, or by applying a baiting bioassay. Based on morphological and cultural characteristics and temperature requirements the following Phytophthora species have been identified: Phytophthora cactorum, P. citrophthora, P. drechsleri, P. cryptogea, hybrid and Pythium. Pathogenicity of the isolates was tested on green apple fruits or one-year-old apple rootstocks. Laboratory studies of the effect of temperature on mycelia growth showed that most isolates can grow from 5° up to 30°C, with an optimum from 18° to 25°C. Only three strains grew at 35–36°C, two developed slowly, one grew well. The optimal pH for mycelia development was tested. Aiming at control of disease, in vivo pot trials have been carried out for studying resistance of rootstocks to P. cactorum. At the end of the growing season a good level of resistance has been shown in the rootstocks M29C, Gizela 6, and MAXMA 14.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Mottle necrosis of sweet potato caused by <Emphasis Type="Italic">Pythium scleroteichum</Emphasis> in Japan and varietal difference in susceptibility to the disease

Severe mottle necrosis (Shirogusare-byo in Japanese) was found on mature tubers of sweet potato (Ipomoea batatas) in Ibaraki Prefecture in October 2004. The causal organism was identified as Pythium scleroteichum hitherto unknown in Japan. Sweet potato cultivar Purple Sweet Lord was more susceptible than cultivars Beniazuma, Benimasari, Koukei-14, and Tamayutaka to the pathogen at 25°C, while this difference in the susceptibility was not clear at 15°C.     

A novel pathosystem to study the interactions between <Emphasis Type="Italic">Lotus japonicus</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

A wilt disease of the model legume Lotus japonicus was observed in a greenhouse in Tokyo, Japan in May 2004. Roots of diseased plants were rotted and dark brown with lesions spreading to lower stems and leaves, resulting in rapid plant death. The causal agent was identified as Fusarium solani based on the morphology. Sequence analysis of rDNA supported the identification. Inoculation of roots of healthy plants with conidia reproduced characteristic disease symptoms, and F. solani was reisolated from lesions, satisfying Koch’s postulates. The isolate also caused chlorotic to necrotic lesions on leaves of healthy plants after wound-inoculation. Infection by F. solani of leaves of L. japonicus was confirmed histologically. Mycelia were observed in the intercellular spaces of parenchymatous tissues in the lesion area and the surrounding tissues. This is the first report of fungal disease on L. japonicus satisfying Koch’s postulates. We named it “Fusarium root rot of L. japonicus” as a new disease. The compatibility of L. japonicus and F. solani is expected to form a novel pathosystem for studying interactions between legumes and fungal pathogens. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB258993 and AB258994.     

Relating plant and pathogen development to optimise fungicide control of phoma stem canker (<Emphasis Type="Italic">Leptosphaeria maculans</Emphasis>) on winter oilseed rape (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">napus</Emphasis>)

In winter oilseed rape experiments at Rothamsted in 2000/01 to 2002/03 growing seasons, the severity of phoma stem canker epidemics in summer depended on the timing of phoma leaf spot epidemics in the previous autumn, and hence on the timing of Leptosphaeria maculans ascospore release. The first major release of L. maculans ascospores was earlier in 2000 (26 September) and 2001 (18 September) than in 2002 (21 October). Consequently, the autumn phoma leaf spot epidemic was also earlier in 2000 and 2001 than in 2002. The resulting stem canker epidemics were severe by harvest (July) in 2001 and 2002 but not in 2003. No correlation was found between the severity or duration of phoma leaf spotting (lesion days or lesion °C-days) and the subsequent severity of phoma stem canker epidemics. Rates of leaf production and loss were similar in the three growing seasons. Out of ca. 25 leaves produced on plants during each season, leaf numbers 10–14 generally remained on plants for the longest. Treatment with flusilazole + carbendazim in autumn decreased the severity of phoma leaf spotting for several weeks after treatment, decreased the severity of stem canker the following summer and increased yield significantly in 2001 and 2002 but not in 2003. The most effective timings for flusilazole + carbendazim application were when leaves 7–11 were present on most plants and at least 10% of plants were affected by phoma leaf spot. Two half-dose applications of fungicide reduced phoma stem canker and increased yield more than a single full dose application when phoma leaf spot epidemics were early (<800 °C-days after sowing).     

Host status and reaction of <Emphasis Type="Italic">Medicago truncatula</Emphasis> accessions to infection by three major pathogens of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

Susceptibility and resistance of <Emphasis Type="Italic">Beta vulgaris</Emphasis> subsp. <Emphasis Type="Italic">maritima</Emphasis> to foliar rub-inoculation with <Emphasis Type="Italic">Beet necrotic yellow vein virus</Emphasis>

Four lines (designated MR0, MR1, MR2, and M8) from 13 accessions of Beta vulgaris subsp. maritima were selected on the basis of phenotypes produced after foliar rub-inoculation with Beet necrotic yellow vein virus (BNYVV). The susceptible phenotype developed bright yellow local lesions, whereas the resistant phenotype had symptoms ranging from no visible lesions to necrotic lesions at the inoculation site. MR1 and MR2 lines had a resistant phenotype depending on the isolate and the MR0 line was susceptible to all isolates of BNYVV tested. The M8 line was highly susceptible; the virus spread systemically and caused severe stunting. These plant lines will be useful for distinguishing BNYVV isolates having different pathogenicities, especially those controlled by RNA3 and/or RNA5.     

Cottony leak of scarlet runner bean caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

Severe cottony leak was found on stems of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture in August 2006. The causal fungus was identified as Pythium aphanidermatum. The name cottony leak of scarlet runner bean (Watagusare-byo in Japanese) is proposed for this new disease.     

Stem and root rot of scarlet runner bean (Phaseolus coccineus) caused by Pythium myriotylum

A severe rot was found on the stems and roots of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture (Japan) in August 2004. The causal fungus was identified as Pythium myriotylum. We propose the name of stem and root rot of scarlet runner bean (“Kuki-negusare-byo” in Japanese) for this new disease.     

Detection and Identification of <Emphasis Type="Italic">Phytophthora</Emphasis> Species in Southern Italy by RFLP and Sequence Analysis of PCR-amplified Nuclear Ribosomal DNA

In four neighbouring regions of southern Italy, Basilicata, Campania, Apulia and Calabria, pepper and zucchini plants showing Phytophthora blight symptoms, tomato plants with either late blight or buckeye rot symptoms, plants of strawberry showing crown rot symptoms and declining clementine trees with root and fruit rot were examined for Phytophthora infections by means of polymerase chain reaction (PCR) assays, using primers directed to nuclear ribosomal DNA (rDNA) repeat sequences. All diseased plants and trees examined tested positive. The detected fungal-like organisms were differentiated and characterized on the basis of primer specificity as well as through extensive restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplified rDNA. Phytophthora capsici was identified in diseased pepper and zucchini plants, P. infestans was identified in tomato with late blight symptoms whereas buckeye rot-affected tomatoes and diseased strawberry plants proved to be infected by P. nicotianae and P. cactorum, respectively. Declining clementine trees were infected with P. citrophthora and P. nicotianae in about the same proportion. Also, thirty-one pure culture-maintained isolates of Phytophthora which had previously been identified in southern Italy by traditional methods but were never examined molecularly, were examined by RFLP and sequence analysis of PCR-amplified nuclear rDNA. Among these, an isolate from gerbera which had previously been identified by traditional methods only at genus level, was assigned to P. tentaculata. For the remaining pure culture-maintained isolates examined, the molecular identification data obtained corresponded with those delineated by traditional methods. Most of the diseases examined were already known to occur in southern Italy but the pathogens were molecularly detected and fully characterized at nuclear rDNA repeat level only from other geographic areas, very often outside Italy. A new disease to southern Italy was the Phytophthora blight of zucchini. This is also the first report on the presence and molecular identification of P. tentaculata from Italy.     

Genetics of host-pathogen interactions in the <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> (net form) – barley (<Emphasis Type="Italic">Hordeum vulgare)</Emphasis> pathosystem

The genetics of host-pathogen interactions in the Hordeum vulgare – P. teres f. teres pathosystem was studied in twelve resistant barley accessions, i.e. CI 9825, CI 9819, Diamond, CI 4922, CI 5401, Harbin, c-8755, c-21849, c-8721 c-23874, c-19979, c-15811. F₂ analyses of crosses with susceptible genotypes employing various isolates (from Europe, USA, Canada, and Australia) revealed that resistance is mostly isolate-specific and controlled by one or two genes. Segregation in ascospore progeny from two crosses between isolates of different origin revealed that avirulence in P. teres is also determined by one or two genes. An epistatic effect of suppressor genes on avirulence genes is proposed for the genetics of virulence to Diamond, Harbin, CI 5401 and c-8721 in the fungal crosses D (181-6 × A80) and F (H-22 × 92-178/9). Segregation in F₂ of crosses of three new sources of resistance (c-23874, c-19979, c-15811) to the susceptible cv. Pirkka was studied in laboratory and greenhouse tests by using seven P. teres isolates, i.e. 181-6, d8-3, d8-4, d9-1, d9-4, F4 and F74. In addition, virulence to these barley accessions of ascospore progeny from crosses of the same isolates was studied. Based on these studies it was concluded that depending on the isolate used, resistance of c-23874 is determined at least by two genes and in c-19979 and c-15811 by three genes. The results of this parallel analyses of genetics of resistance and genetics of virulence allows the postulation of a gene–for–gene interaction in the P. teres – H. vulgare pathosystem.