En tornology FUNGI PARASITIC ON THE NYMPH OF MOGANNZA HEBES WALKER IN TAIWAN L. S; Leu and Z. N. Wang Tainan Sugar Experiment Station Tainan, Taiwan ABSTRACT Two species of parasitic fungi, lsaris sinclairii (Berk.) Lloyd and Metarrhiza anisopfiae (Metsch.) Sorokin, induced mortality in nymphs of Mogannia hebes Walker in sugarcane fields in Taiwan. Nymphs killed by I. sinclairii hardened more than those killed by M. anisopliae. Nymphs killed by both fungi were found in the soil. F. sinclairii could be separated into 2 strains, designated red and white strains, depending on the color of synnemata produced on the dead nymphs. Synnemata were formed by gathering of hyphae and the production of conidia on the periphery. When field-collected nymphs of Mogannia hebes were inoculated by dipping into a conidial suspension prepared from a culture of I. sinclairii, mortality of the nymphs ranged from 39 to 100%. Pathogenicity of this fungus was also shown in a preliminary field test. M. anisopliae, a green muscardine fungus, produced large numbers of olive green spores in conidial masses on the dead nymphs. INTRODUCTION Female adults of the insect Mogannia hebes Walker deposit eggs on the midribs of sugarcane leaves during May and June in Taiwan. After 30-40 days the newly hatched nymphs migrate to the ground, where they remain for 1-2 years feeding on the roots of sugarcane (4). In Taiwan, over 10,000 ha of sugarcane have been infested during the past few years (3). Infestation is believed to cause poor germination and poor growth of ratoon crops (7). Damage to ratoon crops has also been reported to be caused by Melampsalta puer Walker and Parnkalla muelleri Distant in Queensland (9), by Yanga quattalata Signoret in Madagascar (I), and by Mogannia hebes in Ryuku (8). Insecticide applications for control of nymphs of these cicadae appear not to have been successful (1,9). Various parasitic fungi of cicadae are known in nature. Aoki (2) in 1958 mentioned 12 genera of fungi attacking various species of cicadae. An unidentified species of Zsaris was found on the dead nymphs of Mogannia hebes many years ago in Taiwan ancl the phenomenon was reported in 1964 (5). The fungus was reported to produce 14-25y0 mortality. It was believed that once nymphs of Mogannia hebes were attacked by the fungus the insect population declined gradually and finally disappeared from the fields. We began to conduct investigations on the zsaris fungus in 1968 and have experimented with the fungus Metarrhizum anisopliae (Metsch.) since 1970. Previous results with Zsaris sp. have been repqrte'd briefly (6). This paper mainly concerns its culture, pathogenicity, and,identification. Discovery and
542 ENTOMOLOGY observations of the pathogenecity of M. anisopliae are also reported briefly in this paper. MATERIALS AND METHODS Parasitized dead nymphs of Mogannia hebes infected by the red strain of I. sinclairii (Berk.) Lloyd were collected in December 1968 ancl surface sterilized with 1% mercuric chloride solution. The insect bodies were then cut into pieces which were cultured on potato dextrose agar (PDA). After two weeks, conidia of the developing fungus were smeared on PDA and cultured at room temperature (24-28 C) or in incubators set at different temperatures. A thick conidial suspension was then prepared by washing the culture in clistilled water. The white strain of I. sinclairii was collected in 1970 and was isolated and cultured in the same manner as for the red strain. The green powdery residue from dead nymphs was dusted on PDA; then cultures were grown from the isolates obtained on PDA. Inoculation of nymphs was conducted both in pots in the greenhouse and in the field. For pot tests, sugarcane cuttings were planted in 6 inch pots containing non-sterilized garden soil. After the root systems of the cane plants were well developed, fungus-inoculated nymphs were placed 10 cm beneath the soil surface in each pot and covered with additional soil. Nymphs used in the inoculation tests were collected in the field and isolated individually in plastic vials. Mortality among these nymphs was high; however, those living after 24 hrs were dipped for 1-2 min in the coniclial suspension. In field tests, the conidial suspension was dripped into a 10-inch furrow or mixed with sand and placecl in the furrow at the same depth. The conidia were then covered with soil. At regular intervals, a 60 X 60 X 45 cm block of soil was taken from the pots or under sugarcane plants in the field and counts were made of the number of infected nymphs. For cytological studies, nymphs killed by the red strain of I. sinclairii were fixed in formalin-aceto-alcohol, dehydrated with an alcohol series ancl embedded in paraffin. Sections 10 p thick were cut with a rotary microtome, fixed with Haupt's adhesive, stained with safranin and counterstained with fast green. The sections were mounted in Canada balsam for observation. Characteristics of Infected Nymphs RESULTS Newly hatched to mature nymphs were all vulnerable to attack by I. sinclairii and M. anisopliae. Most infected nymphs were found dead in the soil at depths of 15-40 cm, the same depths at which live nymphs were found. So far, no adults have been found to show any symptoms of fungus attack. When nymphs infected by I. sinclairii died, the first unmistakable symptom was hardening of the bodies, followed by the growth of white mycelia over the bodies oi the nymphs. Later, in the red strain angle-like synnemata, 1-2 mm long X 0.5-1.5 mm in diameter with single or several branches, emerged (Fig. I), usually from the head and intersegmented parts of the body. The synnemata later turned brown and became mucoid. Usually a large synnema emerged from
L. S. LEU, Z. N. WANG 543 Fig. 1. Dead nymphs of Mogannia hebes collected from sugarcane fields. Death was inducecl by I. sinclairii. Upper 2 rows with the red strain of I. sinclairii; the lowest row with the white strain. Fig. 2. Dead nymphs of Mogannia hebes 2 weeks after artificial inoculatioll with M. anisopliae. Fig. 3. Structure of the fructificatioll of I. sinclairii. H-hypha; CP-conidiophore; S-sterigma; and C-conidium. Fig. 4. Germination of conidia of I. sinclairii by yeast-like budding 3 days after incubation on the surface of PDA. Fig. 5. Germ tubes of conidia of I. sinclairii. the head of the nymphs. With the white strain of I. sinclairii, fine, rather uniform, coral-like synnemata, about 1 cm long and less than 1 mm in diameter, covered the bodies (Fig. 1). These synnemata remained white and dry. Dead nymphs did not deteriorate in the soil for several months at least. Bodies of nymphs attacked by the green muscardine,fungus did not harden to the degree of those attacked by I. sinclairii. The bodies were covered by a dark olive green powder (Pig. 2). These bodies were found only rarely in the field, probably due to their faster decomposition.
Fig. G-9. Histology of the llymphs of Mogalznia hebes infected wiih I. sinclairii. Histology of Nymphs Killed by Red Strain of I. sinclairii Paraffin sections revealed that the inner part of the body of synnematabearing nymphs was filled with hyphae, and most organs, except fat bodies, were indistinguishable (Fig. 6). The cuticle and fat bodies were stained red and the hyphae were stained green (Fig. 7). Mycelial strands protruded through the cuticle of the nymphs to form synnema (Fig. 8). Conidia in masses were formed around the synnema (Fig. 9). Growth of the Fungi in Vitro The best medium for hyphae growth and sporulation of both strains of I. sinclairii was PDA (Fig 10 and 11). The next best was peanut grain, followed by Sabourad's agar. The fungus grew poorly on autoclaved grains of rice, soybean, wheat, barley, corn and in Czapek's sucrose-nitrate agar. The
L. S. LEU, Z. N. WANG Fig. 10. The red strain of I. sinclairii cultivated on PDA at 26 C. Note the angle-like synnemata. Fig. 11. The white strain of I. sinclairii cultivated on PDA at 26 C. Note the coral-like synnemata. Fig. 12. The green muscardine fungus, M. anisopliae, cultivated on PDA at 26 C. Note conidial masses in green powder. poorest growth was on a fat agar. So far, no sexual stage has been found on any of the media even when they were stored for a year. Optimum temperature for hyphal growth and sporulation was 26-30 C. Sporulation usually occurred 10-20 days after smearing the conidial suspension of FDA or after 3-4 weeks when inoculated on peanut grain. When the fungus was grown on liquid media, growth and sporulation were poorer than on solid media. M. anisopliae has been cultured only on PDA. The fungus grew and sporulated fast, producing a thick, dark green olive powder 6-10 days after it was smeared and incubated at 20-30 C (Fig. 12). Morphological Characters of I. sinclairii The red and white strains of I. sinclairii are similar except in color,
546 ENTOMOLOGY structure, mucilagenous or dryness of synnemata. The hyphae are hyaline, septate and 1-2.5 in width. The conidia are produced singly on slender sterigmata, 1.1-1.9 in length. The conidiophore is single celled, mostly oblong-elliptical. It is hyaline or light yellow, ranging in size from 5.96-9.8 X 2.13-4.26 p. The conidiophore.is flask shaped. It is found singly or in a group as a phialide. Synnema are formed by groups of hyphae. In the red strain, masses of conidia are produced at the periphery from the basal to apical part of synnema (Fig. 3). The fungus obtained from dead nymphs in the field and from inoculated nymphs is similar to that cultured on media. Germination of Conidia of I. sinclairii Conidia of the red strain of I. sinclairii from cultures grown on PDA were centrifuged, washed,twice, then adjusted to log conidia/ml. Germination occurred in 3 days when incubated at 26 C in solutions of potato-dextrose containing a water extract of healthy nymphs of Mogannia hebes and 2y0 glucose. Conidia germinated by extruding 1-3 germ tubes (Fig. 5). Yeast-like budding on the surface of PDA was also observed (Fig. 4). No germination occurred when conidia, were incubated in distilled water and soil extracts. Pathogenicity At regular intervals after inoculation soil in the pots was screened and counts were made of dead nymphs with characteristic symptoms of fungus infection. Throughout the pot experiments, no nymphs in the controls showed symptoms of fungus infection. The results showed that I. sinclairii and M. anisopliae are pathogenic to the nymphs of Mogannia hebes. The time required for development of symptoms on nymphs inoculated with I. sinclairii depended on the month of inoculation. When inoculated in May, only 15 days were required for symptoms to appear and the mortality of the nymphs ranged from 67-10070. On the other hand, inoculation in February required approximately 45 days for symptoms to show and the mortality was 39-83%. In May, the nymphs inoculated with M. anisopliae showed 100yo mortality 2 weeks after inoculation, There was 57-100% recovery of the fungus among isolates from healthyappearing nymphs taken from soil inoculated with I. sinclairii. Field tests showed the mortality of the nymphs ranged from 3-19% during a 2-4 month period after the fungus was inoculated into the soil. No nymphs showed symptoms in the controls. These results suggest that it may be possible to use the fungus artificially to curb field populations of Mogannia hebes. When M. anisopliae was stored at 26 C for 1-3 months, then applied to the soil at the same time that nymphs were placed around the roots of plants in June, it caused 33-10070 mortality. This suggests that the fungus may survive in nature for some time without its host. REFERENCES 1. Anonymous. 1970. La Cigale, Yanga Guttulata. Canne A Sucre Rapport D'Activite 1969, Institue De Recherchos Agronomiques A Madagascar, Document No. 234:237. 2. Aoki, K. 1958. Insect Pathology Gihodo Pub. Co. Toyko, Japan. (in Japanese) 3. Chen, C. B., and T. H. Hung. 19GG. Studies on Cicada, Mogunniu hebes Walker, attack-
L. S. LEU, Z. N. WANG 547 ing on sugarcane ratoon and its control in Taiwan. J. Agr. Assoc. China (New series), 53:31-41. (Chinese, English summary) 4. Chen, C. B., and T. H. Hung. 1968. The cicada, Mogannia hebes Walker, a pest of ratoon sugarcane in Taiwan and its control. Proc. ISSCT, 13:1398-1402. 5. Cheag, W. Y. 1964. The investigation of parasitic disease of Mogannia hebes Walker. Ann. Rep. Taiwan Sugar Exp. Sta. 1963-64. (in Chinese) 6. Leu, L. S., and 2. N. Waag. 1970. The parasitic fungus of Mogannia hebes Walker. Sugar Bull. (Taiwan), 4636-9. (in Chinese) 7. Pan, Y. S., and S. L. Yang. 1968. The nature of injury to sugarcane ratoon caused by the nymph of Mogannia hebes Walker. Proc. ISSCT, 13:1403-1409. 8. Tamoto, S. 1970. Personal communication. 9. Wilson, G. 1968. Cicadas as pests of sugarcane in Queensland. Proc. ISSCT, 13:1410-1415.