Study on etiology and epidemiology of some tulip diseases

I. Study of viral diseases in tulips. 1. Epidemiology of virus-like diseases, mild mottle mosaic and streaking. Two soil-borne virus-like diseases, mild mottle mosaic and streaking, have been found in Toyama prefecture since 1979. Mild mottle mosaic showed color-removing mottle on the flower bud, and color saturation-increasing streak on petals in some cultivars, but did not show any symptom on the flower in whitish and yellowish cultivars. On the leaves, slightly chlorotic mottle mosaics appeared along the leaf veins in most cultivars. White to yellow elongated streaks were seen along the leaf veins, but necrotic veinal streaking or color-banding on petal was rarely seen. Mechanically transmissible agents were isolated from diseased tulips on some diagnosis plants. The agent from the tulips showing mild mottle mosaic symptoms infected 16 species in five families, Chenopodium quinoa, Tetragonia expansa, Nicotiana tabacum cv. White Burley, and N. benthamiana locally. The agent the tulips showing streaking infected 12 species, C. quinoa and Tetragonia expansa locally, and N. tabacum cv. White Barley, N. benthamiana and N. debney systemically. Chenopodium quinoa was a very useful plant diagnosing both diseases, due to the fact that the shape and size of lesions on C. quinoa appeared differently between the two diseases. Maintenance and propagation of mild mottle mosaic agent were successful oh C. quinoa arid other test plants, however, maintenance and propagation for the streaking agent failed on C. quinoa and other plants except systemically infected plants such as N. tabacum White Burley. The mild mottle mosaic and streaking causal agents were mechanically and systemically transmitted from tulip to tulip, and all of the plants exhibited, symptoms in 1991. However, we could not succeed in transmission tests from 1990 to 1993. And, it could not be back-inoculated from C. guinea to tulips. When healthy bulbs were, planted on infested soil with streaking and/or mild mottle mosaic in autumn, the symptoms of mild mottle mosaic were showed in the spring of next year, but the symptoms of streaking were not shown in the spring of next year, and showed typical streak symptom on leaves through progeny bulbs. The percentages of mild mottle mosaic bulb transmission ranged between 2 and 100 % depending on the variety. Transmission percentages for the streaking form were approximately 100 % in most varieties. The incidence of these diseases was decreased when bulbs were planted into the infested soil after mid November or when the pH of infested soil was adjusted to below 5 by adding of sulfur (sublimed). The occurrence of the diseases was suppressed by some fungicidal treatments to soil. Furthermore, the number of resting spores of Olpidium brassicae in tulip roots, was few on the effective treatments. These cases suggested that O. brassicae might' be a vector for these diseases. 2, Characterization of tulip mild mottle mosaic virus. The infectivity of the agent in sap: is very low and extremely unstable. Therefore, we performed our study under optimum conditions for maintaining the infectivity of the extracts, and purified virus-like particles (VLPs) from diseased tulip and inoculated plants and produced an antiserum against the particles. The resulting purified preparations from inoculated C. quirioa, flexuous filamentous VLPs having 4-8nm wide were observed. The particles were observed in partially purified preparations, from naturally infected leaves of tulip and from inoculated leaves of C. amaranticolor, S. oleracea, B vulgaris and T. expansa. VLPs were not observed in healthy leaves. Precipitin reactions specific to crude leaf extracts from infected tulips and C. quinoa were observed in immuno-diffusion tests using the VLPs antiserum. In serologically specific immuno-gold electron microscopy, the filamentous VLPs were decollated specifically with the immunogloblins from VLPs serum. The purified VLP has one protein with an apparent molecular weight of 47 kDa on SDS-PAGE. In western blots analysis, a 47 kDa protein was detected in partially purified preparations made from infected tulips and inoculated C. quinoa, but it was not detected in the healthy control plants. The infectivity in partial purified preparations was completely inhibited by treatment with the immunogloblins from VLP serum (VLP-IgG) at concentrations of 100 and 500micro g/ml, but was not affected by treatment with the antiserum immunogloblins or antiserum against a preparation from healthy C. quinoa (H-IgG). Therefore it seems that the particles are specific to infected plants and are closely related to the mechanical transmission agents isolated from tulips infected with mild mottle mosaic. Treatments with either RNase A, Proteinase K or SDS caused a complete loss of infectivity but DNase I had no effect. When partially purified preparations were subjected to sucrose gradient centrifugation, infectivity from each fraction of preparations was greatly diminished. However, infectivity had increased when certain mixtures of fractions were assayed. Thus infectivity is probably not associated with a single component but with a combination of at least two components which were distinguishable upon sedimentation. A close relationship exists among tulip mild mottle disease, the presence of the associated agent, and the virus-like particles in diseased plants. The associated agent might be multi-partite virus, which contains the 47 kDa protein, and filamentous particles as intact or degraded virion. Considering the results of the experiments using the various preparations, we designed the virus as Tulip mild mottle mosaic virus (TMMMV), a member of genus Ophiovirus. 3. Transmission of mild mottle mosaic by Olpidium brassicae In the transmission experiments using O. brassicae, when tulip plants were exposed to O. brassicae that had been incubated in the roots of virus-infected plants, they contracted mild mottle mosaic. When tulip plants were exposed to O. brassicae that had been incubated in roots of healthy plants, they did on contract the disease. The antigen of tulip mild mottle mosaic virus was detected in all diseased plants by TBIA. The presence of TNV and treatment of hydrochloride with accumulated Olpidium did not influence disease infection rates. These results proved that O. brassicae was a vector of the disease and an antigen of associate particles, the mild mottle mosaic agent was closely related to associate particles, and the agent might be present in resting spores. 4. Properties of other viruses in tulips 1) Tulip breaking virus: TBV TBV particles were purified from infected tulip tissues extracts using clarification with 1/10 vol. of HCP (hydrated calcium phosphate) and 2% Triton X-100. The TBV antiserum had a titer of 1/512 in the ring interface test. Some authors have already reported the symptoms caused by TBV. Furthermore, we observed these kinds of symptoms such as clear breaking, severe breaking, mild (color-added) breaking and color-removed breaking. 2) Lily symptomless virus: LSV A virus isolated from tulips, showing narrow veinal streaks on the petals, was identified as an isolate of lily symptomless virus (LSV) on the basis of host range, particle morphology and serology. LSV was purified from infected tulip leaves, and an antiserum to LSV was produced. The symptoms caused by LSV that appear on the petals, vary depending on the type of cultivar. These symptoms were divided into three types; white streak, color-increasing streak and symptomless. Some of pink colored cultivars showed white streaks. Some red, purple and pink colored cultivars showed color-increasing streak. And all white and yellow colored cultivars and some red, purple and pink colored cultivars were syrnptomless. The symptoms seen on the leaves were those of mild mosaic or symptomless. Triumph, Double Late and Single Late tulips were highly sensitive to LSV. Fosteriana and T. praestance tulips were resistant, although half of the cultivars in Darwin Hybrid showed only moderate resistance to LSV. 3) Tobacco rattle virus: TRV TRV was isolated from infected tulips from some commercial fields. The virus was purified, and an antiserum (TRV-T serum) to the virus was produced. Present three isolates from tulips were reacted with TRV-T serum and an antiserum (TRV-S serum) to a spinach isolate, but not reacted with an antiserum (TRV-N) to a narcissus isolate. 5. Serological diagnosis of virus diseases in tulips. Enzyme-linked immunosorbent assay (ELISA) and tissue blot immunoassay (TBIA), were used to detect viruses in tulips. The successful results were obtained for TBV, LSV and CMV using the standard das-ELISA procedure. However, it was necessary to use Tris-HCl (0.05 M, pH 8.0) or carbonate buffer (0.05 M, pH 9.6), containing 0.1 % 2-mercaptoethanol, 2 % PVP, 0.5 % BSA and 5 % activated charcoal as an extraction buffer for detection of TMMMV by das-ELISA. On the other hand, all of the above viruses were easily detected in tulips standard TBIA procedure. TBV or LSV were detected uniformly within tulip bulbs tested using TBIA, while that of TMMMV was detected locally within tulip bulbs using both methods. Furthermore, TMMMV was presented vertically in tissues of stems and leaves of tulips. II. Study of bacterial post-harvest diseases on tulip bulbs. 1. Identification of the pathogens. Post-harvest blackish-brown rot in tulip bulbs, seemingly caused by bacteria, was found in Toyama Prefecture. The brownish, non-uniform lesions occurred on the outer scales of affected bulbs and were sometimes observed on the top of the second and third scales. Detailed observation of the disease in affected bulbs revealed two different types of symptoms (type-I. -II)- The area of type-I lesions generally spread into the surface tissue of scales, whereas those of type-II generally spread into the inner tissues of the scale. Frequently, the color of the lesions of type-II was more brownish than that of type-I. Causal bacteria were isolated from the affected bulbs. The isolates from type-I lesions were identified as Burkholderia andropogonis, the causal bacterium of bacterial black rot in tulip bulbs, while the isolates from type-II lesions were founded to be Burkholderia gladioli. The disease caused by B. gladioli named as bacterial brown rot. 2. Resistance of tulips to bacterial post-harvest diseases. The susceptibilities to bacterial black rot and bacterial brown rot were investigated using artificial inoculation with 42 tulip cultivars. Most of cultivars were susceptible to bacterial black rot, although severity of the disease varied among different cultivars. On the other hand, about half of the cultivars were distinctively resistant to bacteria brown rot. The severity of both diseases was influenced by several factors, such as principal susceptibility of scale, resulted lesion size, mechanical damage resistance closely related to hardness and field split of bulb coat. 3. Selective medium of B. gladioli. A selective medium (SMG-5) was developed for isolation of Burkholderia gladioli, the causal agent of bacterial brown rot of tulips, from infested soil. Efficiencies of plating of B. gladioli on SMG-5 equal with that of King B medium. Growth of soil bacteria was strongly inhibited on SMG-5 medium. It was difficult to distinguish pathovars of B. gladioli on SMG-5. 4. Epidemiology of bacterial post-harvest diseases. Factors influencing the occurrence of bacterial post-harvest diseases in tulips, caused by B. andropogonis and B. gladioli, during bulb maintenance after lifting were examined. To promote increase the infection rate, it was necessary to wash the bulbs in running water or dip bulbs in bulb mite insecticide. The infection rate increased with number of times the insecticide dip was repeated, and the population of B. gladioli in the insecticide increased. Also, the population of B. gladioli in the insecticides was higher when debris was present with seed-bulb and roots than when debris was removed before washing. The infection rates were influenced by physical damage, wetness, inoculum density and storage temperature. 5. Interaction between pathogens of post-harvest diseases. Infection rates for three post-harvest diseases, bulb rot caused by Fusarium oxysporum f.sp. tulipae, bacterial black rot caused by B. andropogonis and bacterial brown rot caused by B. gladioli, were affected by inoculation with B. andropogonis or B. gladioli. The disease development of bulb rot and bacterial brown rot was increased by the inoculation with B. andropogonis. Those of bulb rot and bacterial black rot were decreased somewhat by the inoculation of B. gladioli. In particular, the promotion of bulb rot by P. andropogonis and the suppression of bacterial black rot by B. gladioli were reproducible. 6. Biological and chemical control of bacterial post-harvest diseases on tulip bulbs. Bacterial black rot and brown rot were reduced by post-harvest treatment with some strains of B. gladioli and B. cepacia isolated from rhizosphere of tulips and welsh onions. While, treatment with B. gladioli strain BRA4, the pathogen of bacterial brown rot, showed a larger reduction of infection of bacterial black rot than that with antagonists from the rhizosphere. Therefore, some weak- or non- pathogenic mutants were obtained from a wild strain BRA4 by NTG treatment or UV irradiation. A mutant-strain N74 showed a highly protective ability against bacterial black rot. Also, post-harvest dipping of bulb in copper fungicide seemed to be effective controlling both bacterial diseases. III. Studies of Botrytis blight in tulips. The sclerotia of Botrytis tulipae germinated two ways, myceliogenic or sporogenic. Mycerial germination occurred underground, and infected to all underground parts of the tulip plant. Sporogenic germination occurred on the soil surface, and sclerotia germinated to produce conidiophores. Sclerotia survived the summer in the field, but did not survive in a paddy field. In Toyama prefecture, most of tulip cultivation is done in fields that are also used for wet-rice cultivation. So, the infested bulbs with the fungus were most important primary inoculum source of botrytis blight in the fields. In past 6 years (1992-97), there has been some close correlation between meteorological conditions and disease epidemics. The severity of the disease in late-April effected by the rainfall and temperature in late-March to early-April, and that at May correlated with rainfall of late-April to May. Regression models were developed to forecast the progress of botrytis blight in fields. The models confirmed the field data.