This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ann, P.-J. Articles by Ko, W.-H. Search for Related Content PubMed Articles by Ann, P.-J. Articles by Ko, W.-H. Agricola Articles by Ann, P.-J. Articles by Ko, W.-H.

Pythiogeton zizaniae, a new species causing basal stalk rot of water bamboo in Taiwan

     Plant Pathology Division, Taiwan Agricultural Research Institute, Wufeng, Taichung, Taiwan

Wen-Hsiung Ko

     Plant Pathology Division, Taiwan Agricultural Research Institute, Wufeng, Taichung, Taiwan, and Department of Plant Protection, National Pingtung University of Science and Technology, Pingtung, Taiwan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

A new species, Pythiogeton zizaniae, was isolated from diseased water bamboo (Zizania latifolia) in central Taiwan. The organism formed a colony with scanty mycelia and mycelial aggregates on rye-water bamboo medium. Special treatments were required for production of sporangia which were terminal, noncaducous and mostly ovoid. Chlamydospores were absent. The fungus was homothallic. Oogonia produced on V-8 water bamboo medium in water were mostly globose to subglobose and each was attached with a club-shaped, monoclinous antheridium by the base of the oogonium stalk. Oospores were plerotic and globose to subglobose. Py. zizaniae caused death of water bamboo suckers but did not infect seedlings of corn, rice, wheat, sorghum, cucumber, tomato, soybean or water spinach. It also did not affect cucumber and tomato fruit, carrot roots or potato tubers.

Key words: Oomycetes, Pythiogeton, water mold, Zizania latifolia

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
The soft swollen stems of water bamboo (Zizania latifolia Turcz.) locally called "Kah-peh-sung" are a popular vegetable in Taiwan. The enlarged portion is a hypertrophy of the first 3–4 nodes beneath the apical growing point, resulting from infection by the endophytic fungus Ustilago esculenta Henn. (Hori 1907, Yang and Leu 1978, Teuell and Batra 1982). This plant, resembling rice, is in the Gramineae family and is planted in flooded fields, developed into clumps each consisting of 20–30 suckers of various ages during the growing season.

In 2000 a serious disease on water bamboo was discovered in the major water bamboo production area in central Taiwan. The outer symptoms consisted of yellowing and browning of young leaves. The internal tissue of the affected basal stems turned brown and became rotten. All plants in the clump eventually died. The disease spread rapidly, and within 1 y it was found in more than 1000 ha of water bamboo farms, representing about 80% of the total production. The disease greatly reduced the yield and caused annual losses of several million U.S. dollars to the growers. A previously undescribed species of pythiaceous fungus was isolated consistently from the diseased plants. The purpose of this study was to characterize the new species of fungus and to confirm its pathogenicity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Isolation and cultivation.— – Diseased tissues of water bamboo with basal stem rot were collected from farms. The fresh diseased stem and shoot tissues were washed in running tap water, cut into pieces (ca. 5 x 5 x 3 mm) and surface sterilized with 0.5% sodium hypochloride solution for 30 sec, blotted dry with paper towel and placed on Petri dish (9 cm diam) containing potato-dextrose agar (PDA) or 1.5% water agar (WA). Each plate contained four pieces of diseased tissues and was incubated at room temperature (23–25 C) for 2–5 d. Fungal hyphal tips growing from the diseased tissues were transferred to rye-water bamboo medium (RWBA) which consisted of 30 g rye-grain extract, 30 g water bamboo swollen stems, 20 g sucrose, 1 g calcium carbonate, 15 g Bacto agar in 1 L medium. Rye extract was prepared by soaking 30 g whole-rye grains in 500 mL of distilled water in a flask for 36 h and heating the flask in boiling water 1 h before filtration through two layers of cheesecloth (Caten and Jinks 1968). Water bamboo juice was prepared by grinding 30 g chopped water bamboo in a blender containing 500 mL distilled water at high speed for 3 min before filtration through two layers of cheesecloth. The two filtrates were mixed and supplemented with sucrose, calcium carbonate and distilled water to make 1 L before autoclave. A substituted medium V-8 juice water bamboo agar medium (V-8WBA, 10% V-8 vegetable juice [Campbell Co.] substituting rye extract) was used for sexual production due to immaturity of sexual organs in RWBA.

Growth and morphology.— – The effect of temperatures on mycelial growth was tested by incubating 3 d old mycelial blocks (0.75 x 0.75 x 0.3 cm) on RWBA plates at 12–40 C with 4 C intervals in the dark. Linear mycelial growth was measured daily for 10 d or until growth reached the margin of the plate. Two plates were used for each treatment and experiments were repeated once.

For sporangium production 10 culture blocks (ca. 3 x 7.5 x 7.5 mm) obtained from culture grown on RWBA at 24 C in the dark for 3 d were placed in a 9 cm Petri plate containing 20 mL of V-8 juice broth consisting of 10% V-8 juice and 0.2% calcium carbonate. After incubation for 3 d under the same conditions V-8 juice broth was decanted and mycelial mats were washed three times with sterile distilled water once every 20 min. Washed mycelial mats were suspended in 20 mL sterile distilled water and incubated at 24 C under a 12 h photoperiod. Morphology of sporangia was photographed under a light microscope and 50 sporangia for each isolate were measured.

For indirect germination of sporangia, mycelial mats with abundant sporangia were incubated at 26 C in the dark for 48 h. The process of development and release of zoospores was observed microscopically.

For sexual reproduction, a small culture block was transferred to a fresh V-8WBA plate and incubated in the dark at 20–24 C for 14 d. Sexual structures were examined and measured microscopically. For production of oospores, four pieces of 7 d old culture blocks (0.75 x 0.75 cm) was transferred into a 6 cm Petri-dish containing 10 mL of distilled sterile water and incubated another 7 d under the same conditions. Diameters of 50 oogonia, oospores and antheridia were measured for each isolate.

Pathogenicity tests.— – Pathogenicity of the fungus to water bamboo and other crops in Gramineae was conducted in a greenhouse. Three week old cuttings of green shell water bamboo were used. The fresh stem sections (5–10 cm long) with several nodes obtained from a disease-free farm were dipped in a 0.5% sodium hypochlorite solution for 1 min before planting in plastic boxes (45 x 30 x 15 cm) containing vermiculate (No. 4) for 3–5 wk. New suckers (15–30 cm tall) were transferred individually into a plastic cup (8 cm diam x 15 cm tall) containing half cup of vermiculate and 300 mL distilled water and inoculated by placing five culture blocks (1 x 1 x 0.5 cm) with sporangia in the water. Disease development and death of cuttings were recorded daily for 30 d. Cups inoculated with water agar blocks were used as control. Five plants were used for each treatment, and the experiments were repeated five times. Pathogen re-isolation was conducted for all artificially infected cuttings.

Pathogenicity to corn (Zea mays cv. Tainung No. 4), rice (Oryza sativa cv. Tainung No. 67), wheat (Triticum aestivum) and sorghum (Sorghum bicolor) studies began with wound inoculation. The 30–45 d old seedlings cultivated in soil in 9 cm plastic pots were used. Basal stems of the each test plant were wounded with a knife and inoculated with mycelial blocks. Each inoculated pot was put in a bigger plastic box containing water to keep soil wet. Disease development was recorded daily for 1 mo. Pathogenicity of the fungus to cucumber (Cucumis sativus), tomato (Lycopersicum esculentum cv. Nonyou 301), soybean (Glycine max) and water spinach (Ipomoea aquatica) also was tested. One-month-old seedlings or cuttings were wounded and inoculated as described above. Fruits of cucumber and tomato, carrot (Daucus carota) roots and potato (Solanum tuberosum) tubers from supermarket also were tested. After washing with tap water and surface sterilization with 75% alcohol, three holes were made on each plant organ with a sterilized cork borer (7 mm diam). A piece of 7 d old mycelial disk on RWBA was inserted into the hole, which was sealed with parafilm. The inoculated organs were kept 7 d on the laboratory bench. Five replicates were used for each treatment, and experiments were conducted twice.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Isolation and cultivation.— – A previously undescribed species of pythiaceous fungus was detected consistently at all water bamboo farms with plants showing mild to severe degree of basal stem rot. The fungus was isolated from the diseased tissues of basal stem, stem gall, parts of leaf sheath and root system but not from healthy plants.

At the beginning of our investigation into the possible cause of the disease, water agar and PDA were used for isolation. Mycelia of a pythiaceous fungus frequently grew out from the diseased tissues into the media after 24–48 h. However, when transferred to fresh media commonly used for cultivation of fungi, including PDA, V-8 agar, rye agar, the mycelia stopped growing. After numerous trials the organism was cultured on media supplemented with homogenized swollen stem tissue of water bamboo. Renewed mycelial growth also occurred when the inoculum was placed on a piece (10 x 10 x 2 mm) of sterile swollen stem tissue of water bamboo on agar media. Rye-water bamboo agar (RWBA) suitable for mycelial growth and repeated subculture was developed for subsequent study. With this medium a total of 79 isolates of the new pythiaceous fungus was obtained from diseased water bamboo plants collected from 25 farms in 2000 and 2001. However the fungus was difficult to maintain. Attempts to store it in distilled water or liquid nitrogen have not been successful.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Pythiogeton zizaniae P. J. Ann et J. H. Huang, sp. nov. FIGS. 1–12

View larger version (124K): [in this window] [in a new window]   FIGS. 1–12. The morphological characteristics of Pythiogeton zizaniae. 1. Colony of isolate PZ18-1 of P. zizaniae on rye-water bamboo agar (RWBA) plate for 7 d. 2. Smooth, spiral mycelia on agar. 3. Hyphal swelling. 4–6. Spherical or ovoid sporangia produced on mycelial blocks in water. 7–9. Indirect germination of sporangia by releasing protoplasm differentiating into zoospore mass. 10–12. Sexual organs. (iz: immature zoospores; mz: mature zoospores swimming away; os: oogonial stalk; an: antheridium) Bar = 20 µm.

Colonial mycelia scanty, homothallic. Hyphae hyaline, coenocytic, (3–)5(–8) µm diam, smooth, scattered or sometimes aggregated, curved or occasionally spiral, with branches angular <90°, with irregular swellings; chlamydospores absent. Sporangia solitary, terminal or rarely intercalary, papillate, noncaducous, ovoid to infrequently ellipsoidal, (75–) 99(–160) x (46–)63(–110) µm, occasionally bilobulate, transversely attached to sporangiophores near apex. Mature sporangia extruding exit tubes through apex and emitting protoplasm into the vesicles. Zoospores differentiated from released protoplasm in water, ovoid or globose, (20–)25(–30) µm diam, laterally biflagellate. Oogonia terminal or rarely intercalary, globose to subglobose, (40–)66(–100) x (38–)53(–70) µm, with the wall (6–)14(–24) µm. Antheridia club-shaped, (8–)15(–20) x (10–)16 (–22) µm, monoclinous, attaching at base of oogonial stalk. Oospores solitary, plerotic, globose to subglobose, (36–)60(–96) x (34–)47(–62) µm. Cardinal temperatures: (16–)28—32(–36) C for mycelial growth, (20–)24(–36) C for producing sporangia, (24–)26(–27) C for indirect germination and (20–) 20(–24) C for sexual reproduction.

Holotype. – HAST.

Etymology. – zizaniae, referring to the genus name of water bamboo.

Pathogenicity tests. – Pathogenicity of P. zizaniae to water bamboo was confirmed by artificial inoculation followed by successful re-isolation. Abundant zoospores appeared in the water of the inoculated cups at the 2nd or 3rd d after inoculation. All water bamboo suckers inoculated with Py. zizaniae wilted and died within 7 d. All control suckers remained healthy at the end of the experiment. Py. zizaniae was re-isolated from the disease tissues of the inoculated suckers, thus fulfilling Koch’s postulate for proving pathogenicity. The pathogen did not infect the seedlings of corn, rice, wheat, sorghum, cucumber, tomato, soybean or water spinach. It also did not affect cucumber and tomato fruits, carrot roots or potato tubers.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Among the eight species of Pythiogeton reported before our study, only Py. zeae Jee et al (Jee et al 2000) was proved to be a plant pathogen. All other species have bean considered saprophytic by Jee et al (2000), although Py. autossytum Drechsler (Drechsler 1932) was isolated from dying tissues of cattail (Typha latifolia), Py. ramosum Minden (Minden 1916) from beet root and Py. dichotomum Tokunaga (Ito and Tokunaga 1935) from rice plant, pathogenicity evidence was lacking. Py. zizaniae is the second species proven to be pathogenic. Unlike Py. zeae, Py. zizaniae was host specific, infecting only water bamboo. In addition to corn Py. zeae also was strongly pathogenic to tomato fruit and moderately pathogenic to oriental melon fruit and carrot root (Jee et al 2000).

Most reported species of Pythiogeton did not form sex organs or were unable to grow in culture (Jee et al 2000). To date only Py. autossytum (Zebrowska 1976) and Py. zeae (Jee et al 2000) are both culturable and capable of producing sex organs. Like Py. zeae, Py. zizaniae was fastidious and died easily during cultivation. However only the former can be subcultured on V-8 agar. The nutrient requirement for growth of Py. zizaniae was much more stringent . It required nutrients so far known to be present only in host water bamboo. Without supplement with host filtrate or tissues, none of test media, including V-8 agar, can support repeated mycelial growth of the new fungus. The required conditions of host nutrient for two species also were different. Autoclaved bamboo filtrate or tissue blocks were effective in supporting growth of Py. zizaniae, while heated corn extract lost its ability to support Py. zeae (Jee et al 2000).

Morphologically Py. zizaniae can be readily differentiated from Py. zeae and Py. autossystum by its predominantly ovoid sporangia as well as larger oogonia and oospores (TABLE I). Sporangia produced by Py. autossytum (Drechsler 1932, Zebrowska 1976) were bursiform or irregularly saccate, and Py. zeae (Jee et al 2000) were variable in size and shape. Oogonia and oospores produced by Py. zizaniae (40–100 x 38–70 µm) were larger than those produced by Py. zeae (22–72 x 31–52 µm) (Jee et al 2000), which are larger than those produced by Py. autossytum (28–33 µm diam)(Zebrowska 1976). In Py. zizaniae only one antheridium was attached to an oogonium, while in Py. zeae or Py. autossytum one or two antheridia were attached to an oogonium (Jee et al 2000, Zebrowska 1976). In Py. zeae the antheridial attachment was distant from the oogonial stalk base (Jee et al 2000), whereas in Py. zizaniae the antheridium attachment was adjacent the oogonial stalk base. Py. zizaniae also is distinguishable from Py. zeae physiologically (TABLE I). The cardinal temperatures for oospore formation were 20–24 C for the former and 15–30 for the latter. Growth of Py. zeae on artificial medium was slow and erratic (Jee et al 2000), while that of Py. zizaniae was relatively fast on rye-water bamboo agar (<14 mm/d at 28 C).

	ACKNOWLEDGMENTS

 
This research was supported in part by a grant from the Council of Agriculture of Taiwan. We thank Dr Yu-Ming Ju for preparation of Latin diagnosis.

	FOOTNOTES

 
Accepted for publication November 3, 2005.

¹ Corresponding author. E-mail: pjann{at}wufeng.tari.gov.tw

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Caten CE, Jinks JL. 1968. Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation. Can J Bot 46:329–348.

Drechsler C. 1932. A species of Pythiogeton isolated from decaying leaf-sheaths of the common cattail. J Wash Acad Sci 22:421–449.

Ito S, Tokunaga Y. 1935. Notae mycologicae Asiae orientalis I. Trans Sapporo Nat Hist Soc 14:1–33.

Hori S. 1907. On Ustilago esculenta P. Henn. Annal Mycol 5:150–154.

Jee HJ, Ho HH, Cho WD. 2000. Pythiogeton zeae sp. nov. causing root and basal stalk rot of corn in Korea. Mycologia 92:522–527.[CrossRef]

Terrell EE, Batra LR. 1982. Zizania latifolia and Ustilago esculenta, a grass-fungus association. Econ Bot 36: 274–285.

von Minden M. 1916. Beitrage zur biologie und systematik einheimsche submerser Phycomyceten. Falk Mykolog Untersuch Berichte 2:146–255.

Yang HC, Leu LS. 1978. Formation and histopathology of galls induced by Ustilago esculenta in Zizania latifolia. Phytopathology 38:1572–1576.

Zebrowska E. 1976. Nikoflora kilku zbiornikow wodnych Puszczy Kampinoskiej. Acta Mycol 12:77–89.

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ann, P.-J. Articles by Ko, W.-H. Search for Related Content PubMed Articles by Ann, P.-J. Articles by Ko, W.-H. Agricola Articles by Ann, P.-J. Articles by Ko, W.-H.