第26巻 第1/2号 日本線虫学会誌 1996年12月 Growth and Propagation of the Rice Stem Nematode, Ditylenchus angustus, on Rice Seedlings and Fungal Mat of Botrytis cinerea Md. Rustom Au and Nobuyoshi ISHIBASHI* Life cycle studies of Ditylenchus angustus demonstrated that one generation time from egg to egg took 8 days on one-week-old rice seedling at 24-26•Ž. The eggs were laid at 2-celled stage and embryos hatched in sterile water 64-66 hr after the egg- laying. The duration of J2, J3 and J4 was 1,1 and 2 days, respectively. Females started the egg-laying 1 day after the adulthood. Fecundity of D. angustus on rice plant was always higher than on the fungus, Botrytis cinerea. Fecundity declined on both hosts following the pre-culture on the fungi for 1 month (1MF) or for 6 months (6MF) prior to inoculation. D. angustus increased 1067,993 and 734-fold on rice plant 40 days after inoculation with 20 adults (10 females and 10 males) collected from rice plant, 1MF and 6MF, respectively. On B. cinerea after the same period from the same initial population the multiplications were 291 and 229-fold, from the inoculum from rice plant and 6MF, respectively. Jpn. J. Nematol. 26 (1/2) 12-22 (1996). Key words: Ditylenchus angustus , rice stem nematode, life cycle, fecundity. Ditylenchus angustus is an obligate and serious pest of rice plants causing "ufra" disease. Ufra was first reported by BUTLER (4) in 1913 from East Bengal in India, now Bangladesh. Ditylenchus angustus is generally called "rice stem nematode" due to its inhabiting the rice stem. They feed ectoparasitically on younger or soft leaves, leaf sheaths, peduncles and spikelets. As a result, the plants become stunted causing severe yield losses (3). The nematodes are usually spread by water. Ufra is mainly found in deep water rice fields, however, recently it is spreading in irrigated rice fields (2, 15, 17). Severe yield losses of 1.26 to 3.94 t/ha have been recorded with only 4 to 10% initial infected seedlings at the transplanting stage (18). An efficient management of this pest requires information about its life cycle. Recently, growth and reproductive parameters of this nematode have been investigated on fungal host, Botrytis cinerea (1). However, no reports are available about the detailed studies relating to the life cycle and reproduction on its original host rice plant. This paper reports the life cycle and reproduction of D. angustus on the rice plant in comparison with those on the fungus, B. cinerea. MATERIALS AND METHODS A pure culture of D. angustus was obtained from Bangladesh Rice Research Institute, Gazipur, Bangladesh, and was maintained in a glasshouse on a susceptible japonica rice cv. "Reiho" . Nematodes from infected plants were extracted by cutting the upper portion of the stem Department of Applied Biological Sciences, Saga University, Saga 840, JAPAN. *To whom all correspondence should be addressed . ―12― Vol.26 No.1/2 Japanese Journal of Nematology December, 1996 (1 cm. apart). Active nematodes were hand-picked under a stereo microscope and checked by a compound binocular microscope for the establishment of two types of stock culture. Culture on sterile rice seedlings: Ditylenchus angustus was monoxenecally cultured on nutrient medium in a petri dish by using the technique developed by PLOWRIBHT and AKEHURST (20) with a little modification. Briefly, two hulled rice seeds cv. Reiho were surface-sterilized with mercuric chloride (0.1% w/v) for 30 min and rinsed 5 times with sterile distilled water, then placed on GAMBORG's B-5 basal medium (10) supplemented with sucrose (2% w/v) in 9 cm diam. petri dishes with 1% agar. The culture was maintained at 24-26•Ž with 12L: 12D photo period. Thirty days after sowing, the seedlings were inoculated with 30 adult nematodes surface-steril- ized with malachite green (0.1% w/v) for 15 min followed by rinse three times with sterile water. The nematodes were directly pipetted with 15 ill water to a cotton mass wrapped the leaf base of the seedlings. The petri dishes were sealed immediately by polyvinyl chloride tape and returned to the same controlled environment. Culture on the fungal mats of Botrytis cinerea: Botrytis cinerea was inoculated on an oblique substrate in a 18 cm long test tube containing 8 ml potato dextrose agar. Five days after the fungal inoculation, 40 adult nematodes which were surface-sterilized with mercuric chloride (0.01% w/v) for 15 min followed by wash 5 times with sterile distilled water, were introduced to it by pipetting with 20 dal water. The test tubes were incubated at 24-26•Ž in the dark. After the population reached a maximum, the nematodes were subcultured up to 6 months by transferring old fungal mat with nematodes to a new mat. The nematodes multiplied on either sterile rice culture or on fungal mat were used as inocula for the following experiments. 1) Determination of life cycle on rice plant: The inocula used for this experiment were freshly hatched 2nd stage juveniles (J2). To obtain J2 enough for experiment, a large number of eggs was collected from infected rice stems of pre-established sterile rice culture, washed 4 times with sterile distilled water and incubated at 24-26•Ž in a concaved glass slide. The hatched J2 were collected at 4-hr intervals and 15 J2 were inoculated on a one-week-old rice seedling (cv. Reiho). The petri dishes were sealed and incubated under the same condition. The nematodes were recovered at every 24 hr intervals and subjected to gentle heat killing for the record of body size and growth stage. The growth stages were determined on the basis of their body length (11). The investigation continued until the development to adulthood and the commencement of egg-laying. Five seedlings were harvested for each observation day. To determine the time required for egg hatching, the newly laid 2-celled eggs were incubated in sterile water at 24-26•Ž and observed at regular intervals until hatching. 2) Fecundity of D. angustus on rice seedlings from different inoculum sources: Adult nematodes for these experiments were collected from rice plant or fungal mat with two different periods; 1-month-culture on fungus (1MF) or 6-month-culture on fungus (6MF). From rice plants, the nematodes were collected from pre-established nematode population on rice seedlings within 25-30 days after inoculation. Active male and female adults were hand-picked and kept in sterile water under aseptic condition. Ten each of male and female adults with 10,al water were inoculated onto a 30-day old rice seedling growing on B-5 medium by pipetting to a cotton mass stuffed into a sheath crevice. The petri dishes were tightly sealed with polyvinyl chloride tape and returned to 24-26•Ž under 12L: 12D photo period. ―13― 第26巻 第1/2号 日本線虫学会誌 1996年12月 Similarly two separate experiments were conducted with the adults from the stock fungal cultures. of 1MF or 6MF. Active males and females were surface-sterilized in malachite green solution (0.1% w/v) for 15 min and washed four times with sterile water. Ten each of male and female adults were inoculated to a 30-day old rice seedling growing on B-5 medium as mentioned above. The petri dishes were sealed and incubated at 24-26•Ž under 12L: 12D photo period. Nematodes were separately recovered from stem (leaf sheath) and leaf blade 10, 20, 30 and 40 days after inoculation. Nematodes on petri dish lid and on agar surface were carefully collected and counted. Number of nematodes/g wet rice tissue was calculated. Ten replications were maintained for each treatment. From the initial and final population, some of the primary characteristics such as average body dimensions of females or body colour density (lipids) expressed as shade gradation were recorded. Measurement was made including eggs from each distribution, and percentages of growth stages were calculated. Data were analyzed with analysis of variance (ANOVA) and the means were compared by Duncan's multiple-range test (p=0.05). 3) Fecundity of D. angustus on B. cinerea: Adult nematodes were collected from 6 months old B. cinerea culture (6MF). Ten each of male and female adults were introduced to 5 days old culture of B. cinerea growing on PDA medium (agar 1.5%) in test tubes (18 cm long) or in concaved slides (10 mm diam. 3 mm depth). To prevent desiccation, the slides were kept in sealed petri dishes in which a moist filter paper was provided. The test tubes and the petri dishes were incubated at 24-26•Ž in the dark with 10 replicates for each batch. In a separate experiment adult nematodes were collected from the sterile rice culture . Male and female ten each were inoculated to 5 days old B. cinerea culture growing in test tubes or concaved slides, being incubated in the same way as above mentioned . For each treatment 10 replications were maintained. Ten, 20, 30 and 40 days after inoculation, the nematodes were recovered by rinsing the test tubes or concaved slides with water. The remaining nematodes in the fungal mat or in the agar plate were carefully recovered under a stereo microscope. The nematodes recovered were counted and their stages were recorded. RESULTS Life cycle: One generation time from egg to egg was 8 days on rice seedlings growing in vitro at 24-26•Ž (Table 1). The half period of one generation, 4 days, was devoted to juvenile development: 1, 1 and 2 days for the 2nd, 3rd (J3) and 4th stage juveniles (J4), respectively . The average body length was 419 16 (S.D.),um for J2, 576•}38,um for J3, 720•}64,um for J4, 795•}25,um for male adults, and 840•}15 pm for female adults.