ABSTRACT
Eucalyptus grandis × Eucalyptus urophylla hybrid clone is an economically and ecologically important forest variety and is widely planted in Guangxi, China. Black spot, a newly found disease, occurred nearly 5333.3 hectares in an E. grandis × E. urophylla plantation of Qinlian forest farm (N: 21.866°, E: 108.921°) in Guangxi in October, 2019. Infected plants had lesions of black spots with watery margins on petioles and veins of E. grandis × E. urophylla. The size of spots ranged between 3 to 5 mm in diameter. When lesions expanded to girdle the petioles, wilt and death of leaves was observed, which subsequently affected growth of the trees. To isolate the causal agent, symptomatic plant tissues (leaves and petioles) were collected from two different sites, sampled from five plants each site. In the lab, infected tissues were surface sterilized with 75% ethanol for 10 seconds, then 2% sodium hypochlorite for 120 seconds, and rinsed with sterile distilled water three times. Small segments (5×5 mm) were cut from the margins of the lesions, then placed on potato dextrose agar (PDA) plates. The plates were incubated at 26°C in dark for 7 to 10 days. Fungal isolates YJ1 and YM6 with a similar morphology, which were obtained from 14 of 60 petioles and 19 of 60 veins respectively. These two colonies were initially light orange, then turned to olive brown as time progressed. Conidia were hyaline, smooth, aseptate, ellipsoidal, apex obtuse, and base tapering to flat protruding scar, 16.8 to 26.5µm long, and 6.6 to 10.4 µm wide (n=50). Some conidia had one or two guttules. The morphological characteristics were consistent with the description of Pseudoplagiostoma eucalypti Cheew., M. J. Wingf. & Crous (Cheewangkoon et al. 2010). For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 and T1/Bt2b, respectively (White et al. 1990; O'Donnell et al.1998; Glass and Donaldson 1995). Sequences of the two strains were deposited in GenBank (ITS: MT801070 and MT801071; BT2: MT829072 and MT829073). Phylogenetic tree was constructed with a maximum likelihood method, revealing that YJ1 and YM6 were on the same branch with P. eucalypti. Pathogenicity tests of the two strains were performed on three-month-old E. grandis × E. urophylla seedlings, by inoculating 6 wounded (by stabbing on petioles or veins) leaves of seedlings with mycelial PDA plugs (5 ×5 mm) from the edge of a 10-day old colony of strain YJ1 or YM6. Another 6 leaves were treated in the same manner but with PDA plugs as controls. All treatments were incubated in humidity chambers at 27°C and 80% relative humidity, under ambient light. All experiments were conducted three times. Lesions were observed at the points of inoculation, the petioles or veins turned black on inoculated leaves after 7 days, wilting of the leaves were also observed after 30 days, however the controls remained asymptomatic. Re-isolation was made and the fungus had same morphological measurements as the inoculated fungus, thus completing Koch's postulates. P. eucalypti had been reported as a pathogen of leaf spot on E. robusta in Taiwan island (Wang et al. 2016), leaf and shoot blight on E. pulverulenta in Japan (Inuma et al. 2015). To our knowledge, this is the first report of P. eucalypti affecting E. grandis × E. urophylla in mainland China. This report provides basis for the rational prevention and control of this new disease in the cultivation process of E. grandis × E. urophylla.
ABSTRACT
Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 µm (average 26.07×14.51 µm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and ß-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 µL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 µL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.
ABSTRACT
Kadsura coccinea (Lem.) A. C. Smith, an evergreen liana, is widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown numerous biological activities, such as anti-tumor, anti-HIV, and anti-oxidant (Yang et al. 2020). In June 2019, an anthracnose on K. coccinea was observed in a plantation in Longan (23°03´N, 107°54´E), Guangxi province. Disease incidence was up to 30% in a plantation. Its symptoms began as small brown spots that expanded into nearly circular spots (Fig. 1A). To isolate pathogen, diseased leaves were collected. The leaves were sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 90 s, then rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed into potato dextrose agar (PDA) plates. The plates were incubated in an incubator at 25°C in dark for 2-3 days. Fungal colonies with similar morphology of 27 isolates were isolated from the 30 infected tissues. Six representative isolates (YB1 to YB6) were selected to further study their characterization. Fungal colonies were grayish-white, orange-yellow conidial masses could be observed in colonies (Fig. 1C). The mature conidia were colorless and transparent, elliptical, and single-celled, 13.0-21.0 × 4.0-8.0 µm (average 16.92 × 5.92 µm; n =100) (Fig. 1B). The DNA sequences of ribosomal internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1) and ß-tubulin (TUB2) were amplified by PCR using the primer pairs ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b (Wang et al. 2020), respectively. Sequences were submitted to GenBank (Accession nos. MZ040489 to MZ040494 for ITS, MZ069043 to MZ069048 for GAPDH, MZ069049 to MZ069054 for CAL, MZ069055 to MZ069060 for ACT, MZ069061 to MZ069066 for CHS-1, and MZ069067 to MZ069072 for TUB2). These sequences were 98%-100% identical to that of reference isolates JX010278, JX010019, JX009709, GQ856775, GQ856730, and JX010410 of Colletotrichum siamense CBS 125378 ex-type recorded in GenBank. Phylogenetic analysis of combined ITS, GAPDH, CAL, ACT, CHS-1, TUB2 genes with 16 sequences obtained from GenBank using maximum likelihood method showed that the six isolates clustered with two reference isolates of Colletotrichum siamense as a distinct clade (Fig. 2). Based on morphological characteristics and phylogenetic analysis, six isolates were identified as C. siamense. Pathogenicity tests were performed on young, fully expanded leaves of 1-year seedlings. Every leaf was punctured at 6 points on the right half and 6 points on the left half using a sterile needle. A 10 µl conidial suspension (1×106 conidia/ml) was inoculated on each wound on the left-half leaf and a 10 µl sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 2 days and after removing the bags, plants were maintained in a growth chamber at 28°C, 80% relative humidity, and a 12-h photoperiod. Anthracnose spots formed 2 to 3 days after inoculation, whereas the control leaves remained symptomless. Morphological characters matched the descriptions of C. siamense. The pathogen was previously reported to cause anthracnose on Aloe vera (Azad et al. 2020), postharvest anthracnose in mango (Liu et al. 2017), pod rot in cacao (Serrato-Diaz et al. 2020). To our knowledge, this is the first report of anthracnose on K. coccinea caused by C. siamense in China.
ABSTRACT
BACKGROUND: Near surface air temperature and rainfall are major weather factors affecting forest insect dynamics. The recent developments in remote sensing retrieval and geographic information system spatial analysis techniques enable the utilization of weather factors to significantly enhance forest pest forecasting and warning systems. The current study focused on building forest pest digital data structures as a platform of correlation analysis between weather conditions and forest pest dynamics for better pest forecasting and warning systems using the new technologies. RESULTS: The study dataset contained 3 353 425 small polygons with 174 defined attributes covering 95 counties of Guangxi province of China currently registering 292 forest pest species. Field data acquisition and information transfer systems were established with four software licences that provided 15-fold improvement compared to the systems currently used in China. Nine technical specifications were established including codes of forest districts, pest species and host tree species, and standard practices of forest pest monitoring and information management. Attributes can easily be searched using ArcGIS9.3 and/or the free QGIS2.16 software. CONCLUSIONS: Small polygons with pest relevant attributes are a new tool of precision farming and detailed forest insect pest management that are technologically advanced. © 2017 Society of Chemical Industry.
