Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex (Tc). The chi60 and chi70 chitinase genes are located on the gene cluster encoding Tc toxins. To clarify the insecticidal activity of chitinases and their relationship with Tc toxins, the insecticidal activity of the chitinases was assessed on Helicoverpa armigera. Then, the chi60 and chi70 genes of X. nematophila HB310 were knocked out by the pJQ200SK suicide plasmid knockout system. The insecticidal activity of Tc toxin from the wild-type strain (WT) and mutant strains was carried out. The results demonstrate that Chi60 and Chi70 had an obvious growth inhibition effect against the second instar larvae of H. armigera with growth-inhibiting rates of 81.99% and 90.51%, respectively. Chi70 had a synergistic effect with the insecticidal toxicity of Tc toxins, but Chi60 had no synergistic effect with Tc toxins. After feeding Chi60 and Chi70, the peritrophic membrane of H. armigera became inelastic, was easily broken and leaked blue dextran. The Δchi60, Δchi70 and Δchi60-chi70 mutant strains were successfully screened. The toxicity of Tc toxins from the WT, Δchi60, Δchi70 and Δchi60-chi70 was 196.11 μg/mL, 757.25 μg/mL, 885.74 μg/mL and 20,049.83 μg/mL, respectively. The insecticidal activity of Tc toxins from Δchi60 and Δchi70 was 3.861 and 4.517 times lower than that of Tc toxins from the WT, respectively, while the insecticidal activity of Tc toxins from the Δchi60-chi70 mutant strain almost disappeared. These results indicate that the presence of chi60 and chi70 is indispensable for the toxicity of Tc toxins.

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Foxtail millet (Setaria italica) is planted widely in northern China, especially in Hebei, Shanxi, Shandong, and Henan provinces. Although several diseases reduce production of this important crop species, sheath blight is considered one of the important diseases of foxtail millet in China. Sheath blight is caused by a soil-borne pathogen and is difficult to control. Epidemics are most common at the late growth stage of foxtail millet. In August 2013, an outbreak was recorded in Shijiazhuang city, Hebei, with an incidence of about 60%. Typical disease symptoms consisted of large, irregular lesions with reddish-brown margin and as the disease progressed, the plants lodge. Three representative sheath fragments (each 1 cm long) were collected from diseased plants during that outbreak. The samples were disinfected with 0.5% (v/v) sodium hypochlorite, rinsed with sterile water, placed on a water agar plate, and then incubated at 26°C in the dark for two days. After the hyphae appeared, ~3-mm-long hyphal tips from typical colonies were excised and transferred to potato dextrose agar (PDA) plates. Three isolates were obtained and all showed typical features of Rhizoctonia-like fungus. Each isolate occupied its whole plate within 5 days of incubation at 26°C in the dark, and abundant aerial mycelia were produced. The color of all colonies was first orange, turning a salmon color when the mycelia matured. Orange sclerotia appeared after 2 weeks of incubation. The nuclei were stained with DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) and observed under a fluorescent microscope. The hyphal cells were multinucleate and the mycelia branched at a right angle. For molecular identification of the pathogen, mycelia of each isolate were cultured in potato dextrose broth at 26°C for a week, and genomic DNA was extracted from mycelia and used as a template for PCR amplification. The primers set of ITS1 and ITS4 was used for amplification of rDNA-ITS from these isolates and the amplified rDNA-ITS regions of all isolates (GenBank Accession Nos. KJ765700, KJ765701, and KJ765702, respectively) were 99% identical to other Waitea circinata deposited in GenBank (1,2). To further confirm the pathogenicity of the isolates, freshly collected PDA plugs were inoculated on the lower leaf blades of 8-week-old seedlings of the foxtail millet variety Yugu 1. PDA plugs without the isolate were used as a negative control. Five plants were used for each isolate and negative control. After inoculation, pots were placed together in a moist chamber at 26°C. No symptoms developed on the control plants, while obvious lesions appeared on the sheaths of tested plants at 5 days post inoculation and later the plants were lodging. The fungus was re-isolated from diseased plants and confirmed to be W. circinata based on morphological characteristics and sequence analysis as previously described, completing Koch's postulates. Further, on the basis of morphological tests, pathogenicity assays, and molecular analyses, the pathogen of foxtail millet sheath blight was identified as W. circinata (4). Although Rhizoctonia solani AG-1, AG-4 has been reported in earlier studies as the pathogen causing foxtail millet sheath blight, there has been no previous report of the disease caused by W. circinata (3). To our knowledge, this is the first report of foxtail millet sheath blight caused by W. circinata in China. With the spread of high millet plant density and fertilizer application, this disease may become a major threat to foxtail millet; therefore, W. circinata should be taken into account when designing measures for disease control in foxtail millet. References: (2) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (1) M. Fiers et al. Eur. J. Plant. Pathol. 128:353, 2010. (4) W. D. Gao. Acta Phytopathol. Sinica 17:247, 1987. (3) T. Toda et al. Plant Dis. 89:536, 2005.