A stable Leifsonia xyli subsp. xyli GFP‐tagged strain reveals a new colonization niche in sugarcane tissues

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most economically important diseases of sugarcane worldwide. Because knowledge on the interaction of Lxx with its host at the microscopic level is limited, the development of tools to monitor Lxx during the colonization process could shed new light on the processes that control disease development. In this investigation, a transformation protocol was optimized and a mutant Lxx strain engineered that stably expressed the gfp gene in sugarcane tissues. In vitro, the growth of the mutant did not differ from that of the wild type. Also, plants inoculated with both strains showed comparable growth and development when analysed 180 days after inoculation (dai). Fluorescence microscopy of roots, stalks, meristems and leaf tissues of Lxx‐GFP‐inoculated plants was performed at 180 dai. In the leaves, Lxx‐tagged cells were observed within the xylem vessels as has been described before but, in addition, they were found in a new niche within the host tissues, in the mesophyll and in the bundle sheath cells surrounding the vascular system. This finding indicates that Lxx is able to move from the xylem to the parenchyma of the leaf cells. This first report of an Lxx mutant expressing a heterologous gene revealed that colonization of sugarcane by this pathogen is not limited to the xylem vessels as commonly reported.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

First report of fig mosaic virus infecting common fig (Ficus carica) in Japan

For the first time, fig mosaic virus (FMV) was detected in common fig (Ficus carica) trees in Shimane, Japan. These trees exhibited mosaic, ringspots, or distortion, accompanied by chlorosis on leaves and yellow spots on fruits. Some of the symptomatic trees were infested with the eriophyid mite Aceria ficus. The virus was detected based on RT-PCR, followed by sequencing. The amplified 300 base-pair fragments shared 83.5–91.5% identity with the corresponding region of RNA-dependent RNA polymerase gene of FMV isolates previously reported in Turkey, Iran, and Italy.     

Identification of Petunia hybrida cultivars that diurnally emit floral fragrances

In order to identify genetic resources for breeding fragrant petunias for use as bedding plants, volatile compounds released by day from the flowers of 40 commercial Petunia hybrida cultivars were analyzed using a solid-phase micro-extraction technique coupled with GC–MS. The three cultivars with solid deep-blue flowers that accumulate malvidin in corollas with high tissue pH were found to emit abundant iso-eugenol as the principal floral fragrance. Several other cultivars that emitted considerable amounts of methylbenzoate and/or benzylbenzoate from the flower were also identified. Association between the floral fragrance and the other floral traits such as floral anthocyanin composition and corolla-tissue pH was discussed.     

Uterine disorders diagnosed by ventrotomy in 47 rabbits

The type, ages of occurrence, primary complaints, clinical signs and mortality in forty-seven cases of uterine disorders diagnosed by ventrotomy in rabbits were analyzed retrospectively. Endometrial hyperplasia (29.8%) was most frequently observed, followed by uterine adenocarcinoma (21.3%). Tumorous lesions were seen in 46.8% of the cases. The age of occurrence ranged from two years and two months to seven years and six months, with a peak at four to five years of age. The most common primary complaint was bleeding (62.2%), followed by mammary gland abnormality (12.8%) and increased abdominal circumference (10.6%). Physical examinations revealed mammary gland disorders such as mammary cysts in 31.9% of the cases. Uterine disorders were detected by palpation in 15 out of 32 cases with a primary complaint of bleeding. Ultrasonography showed uterine disorders in 21 out of 24 cases, suggesting that ultrasonography could be useful in the diagnosis of uterine disorders. The outcome seemed to be influenced by physical status rather than malignancy of lesions. The mortality was higher in cases with symptoms such as anorexia, emaciation, severe anemia, and dehydration.     

Tyrosinase inhibitory activity of citrus essential oils

Thirteen kinds of citrus essential oils and their volatile flavor constituents were investigated for tyrosinase inhibitory activity. Eureka lemon, Lisbon lemon, Keraji, and Kiyookadaidai significantly inhibited the oxidation of L-dihydroxy phenylalanine (L-DOPA) by mushroom tyrosinase. Citral and myrcene among volatile flavor constituents of citrus essential oils exhibited tyrosinase inhibitory activities with Ki values of 0.318 and 2.38 mM, respectively. The inhibition kinetics analyzed by a Lineweaver-Burk plot indicated that citral is a noncompetitive inhibitor and myrcene is a competitive inhibitor. These results indicated that citral and myrcene are responsible for the tyrosinase inhibitory activity of citrus essential oils.     

Isolation and purification of a protective protein antigen of Erysipelothrix rhusiopathiae

The rapid growth and high survival rate of Erysipelothrix rhusiopathiae was determined using a culture of the bacterium in tryptic soy broth supplemented with 0.3% Tris-hydroxymethyl aminomethane and 0.1% Tween 80 (TT-TS broth). High concentrations of 64, 66 and 43 kDa proteins, which are associated with protection against E. rhusiopathiae infection in mice, were obtained by alkaline treatment of whole cells using 0.05-1 N NaOH. The supernatant of alkaline treated cells (alkaline extract; AE) was stable at alkaline or neutral pH. However, aggregates appeared at neutral pH in the absence of sodium dodecyl sulphate (SDS). A high yield of 64, 66 and 43 kDa proteins was obtained from strain Agata (serovar 5). The proteins were eluted from gel bands following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the AE from strain Agata and designated P64 and P43. The amounts of P64 and P43 isolated were 0.7 and 0.3 mg/16 g of wet bacteria, respectively. In a mouse protection test, 50% protective doses (PD50) of P64 and P43 were 0.58 and 0.63 microgram, respectively. Upon Western blotting of the AE, both anti-P64 and anti-P43 antibodies reacted with the 64 and 43 kDa proteins. From these results, it is suggested that P64 is the most effective protective antigen and that P43 (43 kDa protein) is a degradation product of P64. Therefore, the 64 kDa structural proteins are associated with the induction of a protective activity against E. rhusiopathiae infection in mice.     

Cashew nut shell liquid potentially mitigates methane emission from the feces of Thai native ruminant livestock by modifying fecal microbiota

The methane-mitigating potency of cashew nutshell liquid (CNSL) was evaluated by investigating gas production from batch cultures using feces from Thai native ruminants that had been incubated for different periods. Feces was obtained from four Thai native cattle and four swamp buffaloes reared under practical feeding conditions at the Kasetsart University farm, Thailand. Fecal slurry from the same farm was also included in the analysis. CNSL addition successfully suppressed the methane production potential of feces from both ruminants by shifting short chain fatty acid profiles towards propionate production. Methane mitigation continued for almost 150 days, although the degree of mitigation was more apparent from Day 0 to Day 30. Bacterial and archaeal community shifts with CNSL addition were observed in feces from both ruminants; specifically, Bacteroides increased, whereas Lachnospiraceae and Ruminococcaceae decreased in feces to which CNSL was added. Fecal slurry did not show marked changes in gas production with CNSL addition. The findings showed that the addition of CNSL to the feces of ruminants native to the Southeast Asian region can suppress methane emission. Because CNSL can be easily obtained as a byproduct of the local cashew industry in this region, its on-site application might be ideal.