Mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are browning of vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the symptoms of bacterial wilt disease caused by Ralstonia solanacearum, symptoms of MWD generally started from the bottom of the plants and moved upward. In inoculation experiments, four selected MWD strains caused mulberry shoot leaf wilt, discoloration, and defoliation. They also induced whole plant leaf wilt, defoliation and dark brown discoloration of vascular tissue. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB genes four MWD strains were identified as members of the genus Enterobacter. The 16S rDNA and rpoB gene sequences revealed a close relationship among two isolates, R2-2 and R6-2, and the E. asburiae type strain JCM6051. The isolates showed >98% similarity to E. asburiae JCM6051 in their rpoB gene. These results indicated that isolates R2-2 and R6-2 belonged to E. asburiae. No similarity in 16S rDNA sequences above 97% was found between either of the remaining isolates, R11-2 or R18-2, and any recognized Enterobacter species, suggesting that the two isolates may represent novel Enterobacter species. rpoB gene similarity values between the isolates and Enterobacter spp. type strains were <98%, providing further evidence that the two isolates may represent a novel species within the Enterobacter. The causal agent for MWD was previously reported to be E. cloacae, however, this study found that other Enterobacter spp. (E. asburiae and Enterobacter sp.) also cause MWD.

Phytoplasmas that infect gramineous plants, including Napier grass stunt, sugarcane whiteleaf, sugarcane grassy shoot, and Bermuda grass whiteleaf, have been classified into two closely related groups, 16SrXI and 16SrXIV, based on the 16S ribosomal RNA (rRNA) gene. Subsequently, phytoplasmas associated with coconut and Areca palm in southern India and Sri Lanka have been added into the 16SrXI group. However, the 16S rRNA gene gives relatively poor resolution between these phytoplasmas. In this study, a new set of universal phytoplasma primers that amplify approximately 1 kb of the leucyl transfer RNA synthetase (leuS) gene have been validated on a broad range of phytoplasma taxonomic groups. These have been used along with partial sequences of the secA gene to clarify the taxonomic classification of 16SrXI and 16SrXIV phytoplasmas. Based on this data, the sugarcane whiteleaf and grassy shoot phytoplasmas appear to be the same phytoplasma. The Napier grass stunt phytoplasma forms a distinct group from the Bermuda grass whiteleaf and sugarcane phytoplasmas, suggesting that Napier grass stunt should be in its own ‘Candidatus Phytoplasma sp.’. The phytoplasmas associated with coconut and arecanut in southern India and Sri Lanka, which are in the same 16SrXI group, appear in different groups based on secA analysis.

In August of 2006, a new bacterial disease was noted in Hangzhou mulberry orchards of Zhejiang Province, China where bacterial wilt of mulberry caused by Ralstonia solanacearum was previously reported (3). In the summer, the disease caused severe wilt, especially on 1- or 2-year-old mulberry plants, that resulted in premature plant death. Leaf wilt symptoms generally started on older leaves at the bottom of the plant and spread to the younger leaves. The leaves of infected plants became withered and dry, turned dark brown, and eventually the plants became defoliated. The root xylem of infected plants was moist and discolored with brown stripes. The phloem was asymptomatic, however, in severe infections, the phloem was decayed. The observation of wilting proceeding from the bottom of the plant to the top distinguishes this disease from bacterial wilt caused by R. solanacearum. Five bacterial strains isolated from infected mulberry plants showed characteristics similar to those of the standard reference strain of Enterobacter cloacae subsp. cloacae IBJ0611from China, but differed from R. solanacearum IBJ35, E. cancerogenus LMG2693T, and E. cloacae subsp. dissolvens LMG2683T from the University of Gent, Belgium in phenotypic tests, including the Biolog Identification System version 4.2 (Biolog Inc., Hayward CA), pathogenicity tests, transmission electron microscopy (TEM,KYKY-1000B, Japan) observation, and gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Company, Newark, DE) with the aerobic bacterial library (TABA50). Isolates were gram negative, facultative anaerobic, rod shaped, 0.3 to 1.0 × 1.0 to 3.0 μm with peritrichous flagella. Colonies on nutrient agar were light yellow, smooth, circular, entire, and convex with no green fluorescent diffusible pigment on King's medium B (3). Weak hypersensitive reaction was observed on tobacco 3 days after inoculation. All five strains were identified as E. cloacae with Biolog similarity of 0.662 to 0.863 and FAMEs similarity of 0.632 to 0.701. Inoculation of 10 6-month-old intact mulberry plants of cv Husang with cell suspensions containing 109 CFU/ml by pinprick at the base of the stem reproduced symptoms observed in natural infections. No symptoms were noted on the two control plants inoculated by the same method but with sterilized distilled water. The bacterium was reisolated from the symptomatic mulberry plants. E. cloacae has been reported from the United States as the cause of internal yellowing of papaya fruits (1) and rhizome rot of edible ginger (2). To our knowledge, this is the first report of mulberry wilt caused by E. cloacae in China. References: (1) K. Nishijima et al. Plant Dis. 71:1029, 1987. (2) K. Nishijima et al. Plant Dis. 88:1318, 2004. (3) L. Xu et al. Acta Phytophylacica. Sin. 34:141, 2007.

Thirty-six pathogenetic bacterial strains were isolated from wilted mulberry plants in Hangzhou, Zhejiang province of China. The six representative strains were confirmed to be involved in more than one Enterobacter species by common bacteriological test, electron microscope observation, hypersensitive reaction, Koch's postulates, physiological and biochemical test, biology, fatty acid methyl esters analysis (FAMEs), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), 16s rRNA sequences analysis, and comparative analysis with 7 type strains and 3 reference strains. This is the first report on mulberry disease caused by Enterobacter spp. in the world providing new evidence on induction of the plant disease in this genus. The results are not only important in the mulberry disease management but also have significant scientific value for further studies of opportunistic human pathogens and environmental strains in Enterobacter.