No abstract

In October 2006, occurrence of a disease on soybean (Glycine max) was observed in Guangdong Province of southern China with symptoms resembling red crown rot (3,4). Reddish brown lesions girdled the basal stems with numerous reddish orange perithecia on the lesion surface. Roots became black and rotted and whole plants wilted and died. More recently, outbreaks of this disease were observed in several counties in Guangdong. Disease incidence reached as much as 80% on cv. Huaxia 3 in some fields, causing severe yield losses. Isolation was made from the edge of lesions on potato dextrose agar at 25°C. The fungus produced white, aerial mycelia and a burnt orange-to-dark brown submerged growth. Conidiophores were borne laterally on a stipe, terminating in a hyaline, globose vesicle measuring 4.0 to 13.0 μm in diameter. After branching from the stipe, the conidiophore continued to develop by forming two to three single-celled branches. These cells might give rise to two or three shorter branches (14.8 to 36.9 μm long) and phialides. The phialides were doliform, nonseptate, and measured 7.4 to 19.7 × 3.7 to 4.9 μm. Conidia were hyaline, cylindrical with one to three septa (mostly three septa), and measured 54.1 to 76.3 × 4.9 to 7.4 μm. Perithecia were orange to red, subglobose to oval or globose, 212.1 to 454.5 μm high, and 111.1 to 333.3 μm wide. Asci were hyaline, clavate, thin walled, long stalked, measured 121.0 to 200.8 × 11.5 to 25.6 μm, and each contained eight ascospores that became aggregated in the upper half of the ascus at maturity. The ascospores were hyaline, fusoid to falcate with one to three septa (mostly with one septum), constricted slightly at the septum, and measured 29.5 to 73.8 × 4.9 to 9.8 μm. The fungus was identified as Cylindrocladium parasiticum (teleomorph Calonectria ilicicola) (1,2). The beta-tubulin gene fragment sequences of three isolates were obtained (one sequence being GenBank Accession No. GU073284) and comparisons with GenBank showed 99 to 100% similarity with Calonectria ilicicola (EF159730 and AY725643). Pathogenicity was confirmed by inoculation of 2-week-old seedlings of cv. Huaxia 3 in plastic pots (10 × 9 cm) by drenching with a conidia suspension (105 conidia per ml). All inoculated plants showed similar red crown rot symptoms on stem bases and roots 1 week after inoculation. C. parasiticum was reisolated from the diseased plants, and many orange-to-red perithecia of Calonectria ilicicola were formed on the lesions 3 weeks after inoculation. This pathogen may pose a serious threat to >300,000 ha of soybean production as well as >300,000 ha of peanut production in Guangdong Province. It has been previously reported in Jiangsu Province in eastern China (3) and Yunnan Province in western China (4). References: (1) D. K. Bell, and E. K. Sobers. Phytopathology 56:1361, 1966. (2) P. W. Crous et al. Mycol. Res. 97:889, 1993. (3) J. Y. Gai et al. Soybean Sci. (in Chinese) 11:113, 1992. (4) Z. H. Ma et al. Plant Pathol. 53:537, 2004.

In June 2009, disease symptoms on peanut (Arachis hypogaea) were observed in several fields in Huoping county, Guangdong Province. The characteristic symptoms were black rot of the basal stems and roots, with many orange-brown fruiting bodies on the diseased parts. Entire vines eventually wilted and died. The disease incidence reached as much as 30% in some fields, causing severe yield losses. A fungus was consistently isolated from the edge of lesions and grown on potato dextrose agar at 25°C. Mycelia were white and floccose. Conidia were cylindrical to oblong-ellipsoidal, hyaline, one-celled, and measured 3-15 · 1-5 lm. Perithecia were glabrous apart from a number of rhizoidal hyphae, ostiolate and with a neck. The asci were cylindrical, thin-walled, stalk 5-27 lm long, 101-161 lm tall and 10-15 lm in diameter, without discernible apical structures, not evanescent, eight-spored. Ascospores were uniseriately arranged, pale, globose to ellipsoidal, and 7-16 · 7-12 lm.

In July 2010, a serious disease of peanut (Arachis hypogaea) resembling Cylindrocladium black rot (CBR) was found in Longnan County, Jiangxi Province, China. Symptoms included chlorotic, yellowish and blighted leaves, and wilting of the plants. Taproots and hypocotyls were blackened and rotted. Clusters of reddish orange spherical fruiting bodies appeared in the lesions present on basal stems, pegs, pods, and roots of peanut. Disease incidence reached as much as 50% in some patches of the field. Plants with symptoms were sampled from fields. Microscopic examination revealed that the reddish orange, spherical fruiting bodies were the perithecia and measured 461.6 (337.5 to 609.4) × 395.5 (309.4 to 496.9) μm. With gentle pressure, asci and ascospores were exuded from perithecia. The asci were hyaline, thin walled, and long stalked. Ascospores were hyaline, falcate with one septum, and measured 43.5 (27.3 to 54.5) × 5.6 (4.1 to 6.8) μm with a length/width (L/W) ratio of 7.8 ± 1.3. A fungus with white-to-pale buff border mycelia and yellowish brown pigment was consistently isolated from the edge of basal stem lesions on potato dextrose agar at 25°C. Mycelia grew at temperatures ranging from 8 to 32°C and the optimum was 25 to 26°C. To determine the species, single-conidial isolates of the fungus were cultured on carnation leaf agar for 7 days at 25°C and 12 h of light/dark conditions. Conidia were hyaline, cylindrical with one to three septa (mostly three septa), and measured 49.3 (27.3 to 70.9) × 5.9 (4.1 to 6.8) μm with L/W ratio of 8.4 ± 1.6. Vesicles were globose and measured 5.5 to 10.9 μm in diameter. The fungus was identified as Cylindrocladium parasiticum (teleomorph Calonectria ilicicola) (1,2). A PCR assay was conducted on one representative isolate (JXLN32) by analyzing multilocus sequences of the TUB2 (coding β-tubulin protein), ACT (coding actin), and CaM gene (coding calmodulin protein) and were amplified and sequenced using the primers reported by Crous et al. (3). Sequences of the studied DNA regions were submitted to GenBank (Accession Nos. TUB2: JF429649; ACT: JQ070809; and CaM: JQ070808). BLAST searches with the existing sequences in GenBank showed that there was 99 to 100% identity with the existing sequences of C. ilicicola (GenBank Accession Nos. TUB2: AY725643; ACT: GQ280446; and CaM: GQ267402). To complete Koch's postulates, inoculum was prepared by mixing the microsclerotia (MS) suspension of the isolate (JXLN32) with soil at a proportion of 10 MS per g of soil. Ten replicate plastic pots containing five peanut seeds (cv. Yueyou 7) each were planted and placed in a glasshouse at 25 ± 2°C. The same number of peanut seeds was used as an uninoculated control. Typical basal stem and roots rot symptoms of CBR were observed in 2 months and C. parasiticum was reisolated from these inoculated diseased plants. No symptoms were detected on the control plants. To our knowledge, this is the first finding of Cylindrocladium black rot in Jiangxi Province, which is the main peanut-producing area in China. The disease has been previously reported in Guangdong Province in southern China but is not known elsewhere (4). Because of its ability to spread through seed and soil and its destructive potential, this pathogen may pose a serious threat to peanut production in China. References: (1) D. K. Bell and E. K. Sobers. Phytopathology 56:1361, 1966. (2) P. W. Crous et al. Mycol. Res. 97:889, 1993. (3) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (4) R. Pan et al. Plant Pathol. 58:1176, 2009.

to that of the l6SrII-A subgroup. Taken together, these results indicated that the phytoplasma infecting purple coneflower in Taiwan is a 'Ca. Phytoplasma australasiae'-related strain and belongs to the 16SrII-A subgroup. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing WB disease on purple coneflower in Taiwan. The occurrence of phytoplasma on purple coneflower could have direct implication for the economically important ornamental, medicinal plant, and floral industry in Taiwan, especially to the growers and breeders that eagerly promote the purple coneflower industry.