Studies on the epidemiology and control of soilborne diseases of Chinese chive and ginger in Kochi Prefecture, Japan

Kochi Prefecture is the largest producer of Chinese chive (Allium tuberosum) and ginger (Zingiber officinale Rosc.) in Japan. Chronic infections of Chinese chive with soil-borne diseases such as basal rot and pink root rot, and of ginger with Phytophthora rot and Myrothecium rhizome spot have become a major concern because they lead to reductions in yield and quality. Accordingly, the present study was conducted to clarify the epidemiology of these soil-borne diseases and to establish techniques for their control. 1. Basal rot of Chinese chive 1) Occurrence and symptoms Basal rot of Chinese chive, a disease characterized by brown discoloration of the stem plate, shriveling, yellowing, withering, and death of the aerial part of the plant, is widespread in Kochi Prefecture. 2) Identification of pathogenic Fusarium species and their characteristics Two species of Fusarium were shown to be responsible for basal rot of Chinese chive. (1) F. oxysporum . F. oxysporum Chinese chive isolates were virulent to Chinese chive (Allium tuberosum), Welsh onion (Allium fistulosum), onion (Allium cepa), and asparagus (Asparagus officinalis), but not to cucumber (Cucumis sativus), kidney bean (Phaseolus vulgaris), okra (Abelmoschus esculentus), cabbage (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), eggplant (Solanum melongena), sweet pepper (Capsicum annuum), and carrot-(Daucus carota). The host range seems identical with that of F. oxysporum f. sp. cepae, causing basal rot of onion and Fusarium wilt of Welsh onion. From these results, F. oxysporum Chinese chive isolates is determined to be f. sp. cepae (Hanz.) Snyd. and Hans. . Molecular phylogenetic analysis based on the sequences of the rDNA-IGS region revealed that F. oxysporum Chinese chive isolates virulent to Chinese chive were classified at least 3 phyletics. . Of eight Chinese chive varieties, 'Tough Boy' showed resistance to F. oxysporum. (2) F. proliferatum . The Fusarium spp. isolated from Chinese chive plants with symptoms of basal rot, were pathogenic to Chinese chive and different from F. oxysporum in morphology. . The Fusarium spp. isolates formed whitish to light reddish purple colonies on potato dextrose ager (PDA) at 25degC in the dark. Aerial mycelia were white with a cottony texture. The isolates produced microconidia and macroconidia. Microconidia were clavate, pyriform, and mostly unicellular, adhering to conidiophores in long chains and false heads. Conidiophores proliferated inbranched conidiogenous cells that terminated in mono- or polyphialides. Macroconidia were falcate with a foot-cell, up to 5-septate. Colonies grew on PDA at 10-38degC. The optimal growth temperature was 28degC, and the mycelial growth rate was 5.1 mm/day in the dark. . Nucleotide sequences of rDNA-ITS region of the isolates were 100 % identical with the F. proliferatum in the DDBJ/EMBL/GenBank databases, and with the approved isolates of F. proliferatum in the database of NIAS Genebank. The isolates formed a well-supported monophyletic group in neighbor-joining tree for the combined three regions (rDNA-IGS, EF1 sup(alpha), and histone H3), together with the isolates of F. proliferatum reported previous. . Based on these results, the Fusarium spp. Chinese chive isolates were identified as F. proliferatum (Matsushima) Nirenberg. And we therefore propose F. proliferatum as an additional causal agent of basal rot of Chinese chive. . F. proliferatum Chinese chive isolates were virulent to Chinese chive (Allium tuberosum), Welsh onion (Allium fistulosum), onion (Allium cepa), asparagus (Asparagus officinalis), and garlic (Allium sativum) but not to cucumber (Cucumis sativus), kidney bean (Phaseolus vulgaris), okra (Abelmoschus esculentus), cabbage (Brassica oleracea), lettuce (Lactuca saliva), spinach (Spinacia oleracea), eggplant (Solanum melongena), sweet pepper (Capsicum annuum), and carrot (Daucus carota). . One of eight Chinese chive varieties, 'Tough Boy' showed resistance to F. proliferatum. 3) Phylogenetic analysis based on the nucleotide sequences of rDNA-IGS, Histone H3, and EF1 sup(alpha) regions (1) Comparison of molecular phylogeny between pathogens causing basal rot of Chinese chive and those causing Fusarium wilt of Welsh onion . The F. oxysporum f. sp. cepae responsible for basal rot of Chinese chive and that responsible for Fusarium wilt of Welsh onion formed four clusters in the phylogenetic tree constructed based on the sequences of the rDNA-IGS, EF1 sup(alpha), and histone H3 regions. Two of the clusters included both F. oxysporum f. sp. cepae responsible for basal rot of Chinese chive and Fusarium wilt of Welsh onion. . The F. proliferatum responsible for basal rot of Chinese chive and that reported to cause Fusarium wilt of Welsh onion were closely related in terms of the molecular phylogeny. (2) Comparison of molecular phylogeny between pathogens causing basal rot of Chinese chive and nonpathogenic isolates For both F. oxysporum f. sp. cepae and F. proliferatum, pathogenic and nonpathogenic isolates were closely related in terms of the molecular phylogeny. 4) Mating type genes (MAT genes) Both MAT1-1 and MAT1-2 mating types existed in F. oxysporum f. sp. cepae and F. proliferatum responsible for Chinese chive basal rot and Welsh onion Fusarium wilt, and nonpathogenic isolates. 5) The avirulence gene (SIX gene) F. oxysporum f. sp. cepae and F. proliferatum responsible for Chinese chive basal rot carried none of the known avirulence genes (SIX 1-7). Neither F. oxysporum f. sp. cepae and F. proliferatum responsible for Welsh onion Fusarium wilt nor nonpathogenic isolates carried any of the avirulence genes (SIX 1-7). 6) Distribution of pathogens in Chinese chive plant When seeds of Chinese chive were sown in soil contaminated with Nit mutants pathogens of Chinese chive basal rot, the mutants were detected from the basal part (=base of the bulb) in high probability, although no brown discoloration inside the bulb or dry rot was observed during the seedling growth period. When seedling of Chinese chive were planted in a soil contaminated with Nit mutants, the mutants were already detected from the basal part about one month after planting. These results suggest that the base of the bulb is most suitable for isolation of pathogens from Chinese chive plants regard less of growing stage. 7) Simple diagnosis technique Pathogenicity of isolates could be evaluated by a simple inoculation test in which young seedlings of Chinese chive grown in a small glass test tube were inoculated with 1ml of conidial suspension in a concentration of 1.0 * 10E7bud cell/ml. Seedlings showed visible symptom within 7-14 days after inoculation if the isolate was pathogenic. 8) Extinction temperature The infested soil with F. oxysporum f. sp. cepae was disinfested by treating with hot water of 40degC for 168h, 45degC for 72h or 50degC for 12h. The infested soil with F. proliferatum was disinfested by treating with hot water of 40degC for 168h, 45degC for 72h or 50degC for 1h. 9) Control method Soil sterilization by solar heating was effective in controlling the disease. Formigation with dazomet or methamsodium was effective in controlling the disease. Drenching with benomyl, tiophanate-methyl, hydroxyisxazole, validamycinn and phosphorous acid fertilizer during the growth period were also effective in controlling the disease, although no clear differences in efficacy were observed among these treatments. On the other hand, fumigation with 70% chloropicrin tablets and 55% chloropicrin tape against the disease were not effective. 2. Pink root rot of Chinese chive 1) Occurrence and symptoms Pink root rot of Chinese chive, a disease caused by Pyrenochaeta terrestris characterized by pink discoloration in roots and bulb and slight stunting, was found widely in Kochi Prefecture. Sometimes both P. terrestris and F. oxysporum f. sp. cepae or F. proliferatum, pathogens of Chinese chive basal rot, were isolated from the same Chinese chive plant. 2) Temperature for Mycelial growth and disease development Mycelial growth of the pathogen on PDA was observed from 0degC to 35degC with maximum growth at 28degC. Minimum, optium and maximum temperature for disease development is 15, 25 and 30degC respectively. 3) Host range P. terrestris was virulent to welsh onion (Allium fistulosum), tomato (Lycopersicon esculentum), okra (Abelmoschus esculentus), cucumber (Cucumis sativus), kidney bean (Phaseolus vulgaris), Japanese yam (Dioscorea opposita), glory lily (Gloriosa spp.), ginger (Zingiber officinale) and mioga (Zingiber mioga). 4) Time of infection and symptoms Pink root rot of Chinese chive occurred irrespective of infection time, whether at seeding or planting in the field, although little difference was observed in the development of the disease depending on the time of infection. 5) Mixed infection with Fusarium and Pyrenochaeta pathogens of basal rot and pink root rot Additional infection with F. oxysporum f. sp. cepae or F. proliferatum, pathogens of Chinese chive basal rot facilitated the development of pink root rot in Chinese chive. 6) Extinction temperature The infested soil with P. terrestris was disinfested by treating with hot water of 45degC for 300min, 50degC for 10min or 55degC for 3min. 7) Control method Soil reduction and solar heating were highly effective in controlling the disease. Removing diseased crop residues improved the efficacy of both treatments. Fumigation with 70% chloropicrin tablets, 55% chloropicrin tape, dazomet or Metham sodium were effective in controlling the disease. Drenching with triflumizole and benomyl during the growth period were also effective in controlling the disease. 3. Phytophthora rot of ginger 1) Occurrence and symptoms Phytophthora rotof ginger, a disease characterized by water-soaked rot on basal pseudostems and brown rot on rhizomes were first observed, then plants developed stem blight in the field. The disease also developed on rhizomes stored at 15degC in the dark. 2) Identification of pathogen A Phytophthora sp. was consistently isolated from the symptomatic lesions and caused the same symptoms after inoculation with the isolates. The identical Phytophthora sp. was then reisolated. White stellate colonies grew on PDA at a minimum temperature of 10degC, optimum of 23degC and maximum of 30degC. Sporangia were ovoid, ellipsoid, globose and distorted (variable) with one or two apices, noncaducous, 30-90 * 20-50 (average 50.0-56.1 * 25.0-32.6) micro m, with a length to breadth ratio of 1.5-1.7: 1. Nucleotide sequence of the r-DNA ITS regions well agreed with those of Phytophthora citrophthora (R. E. Smith and E. H. Smith) Leonian previously reported. Based on these results, the isolate was identified as P. citrophthora. This report is the first of a disease of ginger caused by P. citrophthora, and we proposed the name 'Phytophthora rot' for the disease. 3) Host range P. citrophthora was virulent to four tested plants, including mioga (Zingiber mioga), eggplant (Solanum melongena), cabbage (Brassica oleracea) and kidney bean (Phaseolus vulgaris). The pathogen was not pathogenic to cucumber (Cucumis sativus), netted melon (Cucumis melo), tomato (Lycopersicon esculentum), sweet pepper (Capsicum annuum), Chinese cabbage (Brassica campestris), Chinese chive (Allium tuberosum), and welsh onion (Allium fistulosum). 4) Optimum temperature for disease development Minimum, optium and maximum temperature for disease development is 10degC, 20degC and 30degC, respectively. 5) Time of disease appearance under open field conditions In field observation water-soaked rot on basal pseudostems and rhizomes were observed in early summer (between early June and early July) and autumn (between early October and late November). The disease increased under rainy weather conditions with an average temperature range from 21 to 22degC. 6) Disease development during storage Diseased rhizomes with black discoloration in buds sprouts and white mycelia were found by two months after storage at 15degC. Surfaces of rhizomes turned to brown and were eventually fully covered with white mycelia. The insides of rhizomes also turned into brown, water-soaked appearance and softed gradually. Ultimately, inside of the rhizome decayed leaving only the epidermis. Symptoms developed during strage at 15degC in rhizomes infected before strage. Symptoms also appeared on healthy looking rhizomes derived from diseased stocks within 2 months at 15degC. In rhizomes contaminated with infested soil, symptoms appeared 2-3 months after storage at 15degC. Thus, the damage by Phytophthora rot spread during strage as a result of disease development in infected rhizomes themselves and infection of healthy rhizomes though contact with diseased rhizomes or contaminated soil. 7) Extinction temperature The infested soil with the pathogen was disinfested by treating with hot water of 40degC for 180 min, 45degC for 10min or 50degC for 3min. 8) Control method Soil reduction using wheat bran, flooding and fumigation with amixture of chlorpicrin and 1,3-dichloropropene, dazomet (30kg/10a,60kg/10a) or 99.5% chloropicrin were effective in controlling the disease. Drenching with cyazofamid during the growth period was also effective in controlling the disease. 4. Myrothecium rhizome spot of ginger 1) Occurrence and symptoms Black spots that appeared on the surface of the rhizomes of ginger during strage had led to serious economic losses in Kochi Prefecture, Japan. Microscopic observations of the internal parts of the black spots revealed that certain mycelia were observed but they did not invade into the rhizomes deeply. Rhizomes did not rot at all. 2) Identification of the pathogen A fungus was frequently isolated from the symptomatic rhizomes. In evaluation of pathogenicity, the isolate caused the same symptoms on the ginger rhizomes, and was reisolated. The isolate formed white stellate colonies with a greenish black spore mass on PDA. Mycelia of the isolate on PDA grew from 10degC to 35degC with maximum growth at 30degC. Conidia on sporodochia bearing greenish black spore mass. Conidia produced from phialides on branched conidiophores. Sporangia were fusiform, monocellular, and 7.5-5.0 * 2.0-3.0 (average 6.4 * 2.5) micro m in size. The morphology of the isolate agreed with that of Myrothecium verrucaria (Albertini et Schweinitz) Ditmar. The nucleotide sequence of the r-DNA ITS regions well agreed with those of M. verrucaria. In conclusion, the isolate was identified as M. verrucaria. We proposed to designate the disease as Myrothecium rhizome spot of ginger. 3) Host range The ginger isolate of M. verrucaria was virulent to six tested plants, including Chinese cabbage (Brassica campestris), cabbage (Brassica oleracea), spinach (Spinacia oleracea), okra (Abelmoschus esculentus), Japanese radish (Raphanus sativus) and carrot (Daucus carota), but not to cucumber (Cucumis sativus), netted melon (Cucumis melo), tomato (Lycopersicon esculentum), eggplant (Solanum melongena), sweet pepper (Capsicum annuum), Chinese chive (Allium tuberosum) and welsh onion (Allium fistulosum). 4) Development of the disease The disease developed about one month after storage or later, and the black spots increased in number and thickened as storage periods extended. 5) Evaluation of organic materials as infection sourse The ginger isolate of M. verrucaria formed conidia on three kinds of organic material, 'keintoppu' (sugar cane crop) residues, rice straw and wheat straw used as mulching material for ginger cultivation. 'Keintoppu' may provide a propagation site for pathogen since the pathogen easily formed spore mass on its surfaces. 6) Suppression of black spot formation Fumigation with dazomet or Metham sodium was effective in controlling the disease. Immersion treatments into solution of benomyl, triflumizole, or thiophanate-methyl was effective in suppressing black spot formation caused by Myrothecium verrucaria.