In contrast to other transgenic Bacillus thuringiensis (Bt) crops (e.g. Bt maize and cotton), risk assessments of Bt rice on soil ecosystem are few. To assess the influence of Bt rice on rhizosphere soil ecosystems, soil samples from Bt, non-Bt and controls were taken at seedling, tillering, booting, heading and maturing stages. The activities of dehydrogenases, invertase, phenol oxidases, acid phosphatases, ureases and proteases showed no significant differences between Bt and non-Bt rice. A Biolog system was used to evaluate the effect of Bt rice on functional diversity of microbial communities. Although there were differences in carbon substrate utilization between Bt and non-Bt rice at seedling, tillering and heading stages, these differences were transient and not persistent. Denaturing gradient gel electrophoresis (DGGE) fingerprint patterns showed that Bt rice had little effect on the dominant rhizosphere bacterial, fungal and actinobacterial communities. The richness and consistency of microbial communities according to carbon substrate utilizations and DGGE band patterns did not differ significantly between Bt and nonBt rice, and were close to that of control soil. There was no evidence to indicate apparent effects of Bt rice on soil enzyme activities, microbial community composition and functional diversity in this study.
Guava (Psidium guajava) cv. Watermelon Bar was introduced into Guangxi province of China in 2015, and subsequently planted on more than 5,000 ha in Yulin city alone. Black spot disease on fruit was observed between June and October 2015. Once few diseased fruits were found on a guava tree, more than 30% fruits were found to be infected in a several days later. In the field, the fruits from more than 90% guava trees were infected by the disease. Spots containing pycnidia were dark brown to black, sunken, and 3 to 22 mm in diameter. Nine diseased guava fruits were collected from three trees in West Bank village (22.695387N, 110.112299E), Yuzhou District, Yulin City of Guangxi in China. Tissues from the margin between healthy and diseased portions of the fruit were excised and surface sterilized by immersing in 75% ethanol for 3 min, washed with sterile water three times, cut into 0.2 to 0.3 cm2 pieces, and placed on potato dextrose agar (PDA). After 3 days of incubation at 27℃, greyish-green fungal colonies developed. Hyphal tips were subcultured on PDA and OA (oat agar), incubated at 27℃ in a 12 h light/12 h dark cycle for 9 to 10 days until small black pycnidia appear under the mycelia, after which conidia were observed from pycnidia under the optical microscope. Pycnidia were black, granular and in clusters. Conidia were hyaline, unicellular, obovate, 7.47 to 14.49 μm × 5.01 to 9.63 μm, with mucoid sheath and apical mucilaginous appendage. After incubating for 20 days, black, granular and agglomerate perithecia under the mycelia were observed. Pressed the perithecia slightly on a slide, asci were squeezed out which were 8-spored, subcylindrical to clavate, 56.74 to 88.27 μm × 7.88 to 12.16 μm. Ascospores were hyaline, unicellular, multiguttulate, fusiform, wider in the middle, and 10.50 to 16.19 μm × 3.64 to 5.86 μm. Spermatia were hyaline, bacilliform with swollen ends, 3.15 to 8.99 μm × 1.02 to 1.88 μm. The single-spore isolate, YLWB01, was selected as one of 24 representative isolates, for molecular identification. DNA of the isolate YLWB01 was extracted, and the internal transcribed spacer (ITS) region, and the actin (ACT) and translation elongation factor 1 alpha (TEF) gene fragments were amplified using primers ITS1/ITS4 (White et al. 1990), ACT512F/ACT783R and ER728F/EF986R (Carbone et al. 1999), respectively. BLAST analysis in the GenBank database shown that obtained sequence of ITS (MN958712), ACT (MN958710) and TEF (MN958711) had 100% identify to Phyllosticta capitalensis (GenBank accession: AM403717, JN791558 and FJ538377, respectively). Phylogenetic analysis by neighbor-joining using MEGA 6.0 (Tamura. et al. 2013; Guarnaccia. et al. 2017) results shown that ITS, ACT and TEF sequences clustered together with P. capitalensis (strain CPC18848) in the phylogenetic tree. Based on these morphological characteristics and sequence analysis (Wikee et al. 2013), the fungus was identified as P. capitalensis, cause of guava black spot disease (Arafat 2018). Pathogenicity of YLWB01 single-spore isolate was demonstrated on fruits surface sterilized by 75% ethanol in the laboratory, six symptomless mature guava fruits were stab wounded, three stabs per fruit, then three fruits inoculated with a 5-mm-diameter colonized disk of PDA incubated for 10 days and three fruits inoculated with blank PDA disk as control. After inoculation, the fruits were incubated in glass covers at room temperature. Symptoms began to appear after 2 days and were similar to those of naturally infected guava fruits after 5 days. Control fruits remained healthy. The same fungus was re-isolated from the inoculated fruits, but not control fruits, using the methods described above. Guava black spot disease caused by P. capitalensis has been previously reported in Egypt (Arafat 2018). Zhang et al. (2011) also reported detection of P. capitalensis in China on imported guava fruit with black spots inspected by the Airport Office of Ningbo Entry-Exit Inspection and Quarantine Bureau in Zhejiang province of China, but not collected from the field. To our knowledge, this is the first report that P. capitalensis is the pathogen of guava black spot disease in mainland China.
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