Bacillus subtilis strain IK-1080 was tested for practical use as a biological control agent against rice blast disease. When the anatagonist B. subtilis IK-1080 was cultured with the rice blast fungus on potato sucrose agar plates, hyphal growth was greatly suppressed. Blast fungal spores (103 spores/ml) were added to suspension solutions of B. subtilis IK-1080 at 1.0×106, 5.0×107, 1.0×108 and 5.0×108cfu/ml and left to germinate on cellophane on a water agar plate. The rate of both germination and appressorial formation began to be suppressed at 1.0×108 and 5.0×107cfu/ml of B. subtilis, respectively. At 5.0×108cfu/ml of B. subtilis, germination and appressorial formation (62.3% and 11.2%, respectively) were much lower than those of the controls (96.3% and 56.4%, respectively). When 5-ml suspensions of B. subtilis IK-1080 at the same concentrations as those stated earlier were sprayed on plants at the 4.5-leaf stage of rice cultivar Koshihikari followed by spray inoculation with a suspension of blast spores (1.0×104 spores/ml), leaf blast was significantly suppressed by the antagonist at 5.0×107cfu/ml or higher. The reduction in disease severity was strongly correlated with the suppression of appressorial formation (r=0.9374, p<0.01). When a suspension of B. subtilis IK-1080 was applied to blast disease lesions that had formed on the leaves 14 days after the inoculation with fungal spores, the mean length of the lesions at 7 days after application of the bacteria (13mm) did not differ from that of lesions on control plants without bacteria. However, two applications of a suspension IK-1080 on leaves, before inoculation with fungal spores and 14 days later, greatly reducted mean lesion length (6mm). In a paddy field experiment with yearly outbreaks of blast disease in 2000, a suspension 1.0×108cfu/ml of IK-1080 was sprayed on leaves of rice plants just before heading and again 9 and 18 days after heading. Disease severity was suppressed by more than half. Good results were also obtained (in 2001) in the same paddy field with a suspension of 1.0×108 and 5.0×108cfu/ml of B. subtilis IK-1080 sprayed on leaves of plants just before heading and at 13 days and 23 days after heading. B. subtilis IK-1080 at 1.0×108 and 5.0×108cfu/ml reduced disease seventies to 13.8 and 7.7, respectively, with high protective values of 52.5 and 73.5, respectively.
When 1.0ml of a suspension of B. subtilis IK-1080 at 1.0×108cfu/ml was sprayed onto petals of a tomato plant at the same time as a hormone treatment to enchance fruiting, gray mold disease on petals was greatly suppressed. The number of the bacteria on the petals was 1.9×107cfu/g just after spraying and increased about 400 fold to 7.8×109cfu/g by 17 days after hormone treatment. More than 10 species of filamentous fungi, including species of Botrytis, were found on petals of tomato plants in the untreated plot, but only five were filamentous fungi and none were Botrytis, on petals of tomato plants that had been sprayed with a solution of IK-1080, indicating that this treatment resulted in a decrease in the number of species of fungi on the petals. Bumblebees, often used to pollinate tomato, were tested as a carrier for an inhibitor of gray mold, targeting tomato plant petals. We tested four types of insect vector adapter, three types that were constructed as part of the beehive and one type that could be attached to the entrance of a hive (detachable type). Many of the bees were reluctant to enter the former three types of adapter, and many pollen grains were dropped. In the case of the detachable adapter, both the number of bees leaving the hive per hour and the percentage of bees returning to the hive per hour were high (12 and 77.8%, respectively). Fruiting rates of the tomato plants visited by bees from the hive with the detachable adapter were also high (96-98%). When the detachable adapter was used, 6.0×104cfu of IK-1080 adhered to the body of each bee. The use of bumblebees as vectors to carry IK-1080 resulted in an increase in the number of the IK-1080 bacteria on the petals of tomato plants to 106∼107cfu/g at 20 days after the bees had visited the plants as well as a dramatic reduction in gray mold on petals.
The survival of Acidovorax avenae subsp. citrulli (Aac) on watermelon seeds and the population dynamics of Aac on watermelon seedlings were examined. Aac survived for over 26 months on infested seeds that were stored at 4, 10, 15, 20 and 30°C. These infested seeds were then thought to become a source of infection. When infested seeds with only 1 cfu of Aac were sown and grown under high humidity, there was a high probability of the germinated seedlings becoming diseased. The percentage of diseased plants was higher under high humidity than low. When germinated seedlings were inoculated with Aac (105cfu/ml) by spray inoculation and then grown at high humidity, the population of Aac on watermelon seedlings reached 106-107cfu/g fresh weight from below 10cfu/g fresh weight and seedlings were diseased by 2 days after inoculation. When the inoculated seedlings were incubated at low humidity, Aac multiplied to 104-105cfu/g fresh weight on seedlings by 2 or 3 days. However, no seedlings were diseased. From the data obtained by different isolation methods, Aac grew mainly on the surfaces of the seedlings.
When a wettable powder of Bacillus subtillis IK-1080 (IK-1080) was dusted daily at 10g/1000m2 through ducts of a hot-air heating system for growing cucumber plants in a greenhouse, gray mold did not develop during the experimental period (November 1999 to April 2000). In a conventional fungicide plot, on the other hand, the disease developed on 8.3% of plants by February and on 25.5% of plants by April. Even when the petals on cucumber plants that had been dusted with IK-1080 powder were inoculated with the gray mold fungus under laboratory conditions, colony development of the pathogen was greatly suppressed. When cucumber plants were dusted with IK-1080 powder via the hot-air ducts, the velocity of airflow from the duct outlets was highly correlated with the number of IK-1080 colonies formed on nutrient agar. At 35 days after dusting with IK-1080, the mean concentrations of IK-1080 on upper, middle and lower leaves of the plant were 3.4×104, 1.1×104 and 2.3×103cfu/cm2, respectively. Mean concentration of IK-1080 on petals was remarkably high (2.0×109cfu/g). IK-1080 was reisolated from fruits and stems of plants. IK-1080 was not detected on any leaves of plants in the control plot. Uniform dusting with IK-1080 throughout the greenhouse was possible by setting the main ducts of the heating system around the inside perimeter of the greenhouse and the branch ducts on the ridges between plants, then operating the heating system at an airflow of 10m/s. In the control plot, species of Cladosporium, Alternaria and Mycosphaerella were found on 47-78% of plant petals, and Nigrospora, Fusarium, Botrytis and four unidentified fungi were also detected (8-38%). In the IK-1080 plot, Cladosporium and Alternaria were detected at 54-91%, but only two other unidentified fungi were detected (9-11%). The number of fungal species on petals can be reduced by daily application of IK-1080 through hot-air ducts.
Root tumors developed on melon plants after either pouring inoculum of pathogenic Streptomyces sp. into soil surrounding growing plants or sowing melon seeds in soil inoculated with the pathogen with a latent period of ca. 7-14 days. In melon sown in infested soil, root tumors were first observed on branches of the primary root at 14 days. These tumors then began to form on other branches. Although numbers of root tumors increased until about 42 days after sowing in infested soil, the number of new tumors decreased about 49 days after sowing. By 49 days after sowing, length of infected melon plants were about 60% of that of healthy melon plants and the infected plants had about 85% of the number of leaves on healthy plants. When melon plants were inoculated at different growth stages, the most severe symptoms were observed on plants inoculated when 14 to 21 days old. Severity of the disease was mitigated with aging in plants inoculated after 21 days of growth. Development of symptoms is therefore closely related to the growth stage at the time of inoculation, with formation of root tumors more active when the root system is actively developing. On the other hand, injecting the pathogen into aerial melon parts causes morphological abnormalities on inoculated tissues. Although this pathogenicity was most severe on hypocotyl tissue, abnormalities on stem and petiole were milder than those on hypocotyl and were more conspicuous when plants were inoculated with mycelia than with spores.
A supervised disease management system that halves the number of fungicide applications on Japanese pear was investigated from 1993 to 2001 in experimental and commercial orchards of Chiba Prefecture. The concept for the strategies was based on integrated pest management (IPM). Sterol demethylation inhibitors (DMIs), strobilurins, iminoctadine arbesilate and others were highly effective and had long-lasting activity against pear diseases. The former two fungicides, at high risk for fungicide resistance, were mixed with other fungicides that had different mode of actions. The fungicides were sprayed on a day just before rainfall, and no further application was made as long as a residual effect of the sprayed fungicide was expected. To reduce the source of primary infection, infected, fallen pear leaves were cleared from the ground in winter, and diseased clusters were removed from shoots in early spring. The effectiveness of the system proved to be practical to control major diseases, including scab on leaves and fruit caused by Venturia nashicola and fruit ring rot caused by Botryosphaeria berengeriana. In the commercial pear orchards in this sturdy, fungicides were applied only seven times spring, up to fruit harvest for ‘Kousui’, half of the fifteen officially recommended sprays in Chiba Prefecture in 1993.
A new disease of eggplant was found in greenhouses in Kochi Prefecture, Japan in 1996. Numerous black pycnidia were produced on brown lesions that developed from the cut surface of lateral shoots. Pycnidia were ostiolate, subglobose to pyriform, and averaged 301×248μm. Conidia were born mostly holoblastically on sometimes branched conidiophores, hyaline, aseptate, oblong to fusiform, 13.8-20.0×4.4-5.6 (mean=17.2×5.0)μm, with a truncate base. The fungus was identified as Fusicoccum aesculi on the basis of its characteristics. The symptoms were reproduced by wound inoculation, and the fungus was re-isolated. Stem blight of eggplant was proposed for the name of the new disease.
A new bacterial blight with halo was found on leaves of rye (Secale cereale L.) in Tsukuba, Japan in April 1998. The bacterium isolated from the lesions of the leaves was pathogenic to rye and oats, but not to rice. The causal bacterium was elucidated as a pathovar of Pseudomonas syringae on the basis of bacteriological properties. In addition, tabA-specific bands were detected from these isolates by PCR with tabA-specific primers. From these results, the causal bacterium was identified as Pseudomonas syringae pv. coronafaciens, and the name “bacterial halo blight” was proposed for the disease.