Tobacco (Nicotiana tabacum L.), one of the chief commercial crops, is wildly cultivated worldwide. In June 2020 and 2021, an unknown bacterial leaf spot on tobacco was found in Hezhou and Hechi City, Guangxi, China. 30% of the tobacco were affected and the rate of diseased leaves reached about 10% in the field under high temperature and rainstorm. The disease mainly damaged the middle and top leaves of tobacco plants at vigorous growing stage. The initial symptoms were water-soaked spots on the frontal half of a leaf, and then expanded into circular to irregular spots with a yellow halo at the edge. The spots mostly appeared dark brown at high air humidity, while yellow brown at low humidity and exhibited a concentric pattern. In severe cases, the lesions coalesced and the whole leaf was densely covered with lesions, resulting in the loss of baking value. A bacterium was consistently isolated from diseased leaf tissues on nutrient agar (NA). Growth on NA was predominantly grayish white circular bacterial colonies with smooth margins, and the bacterium is rod-shaped, gram-negative and fluorescent on King’s B medium. Seven isolates (ND04A-ND04C and ZSXF02-ZSXF05) were selected for molecular identification and pathogenicity tests. Genomic DNA of the bacterium was extracted and the housekeeping gene of cts (encoding citrate synthase) was amplified with the primers cts-Fs/cts-Rs (forward primer cts-Fs: 5’-CCCGTCGAGCTGCCAATWCTGA-3’; reverse primer cts-Rs: 5’-ATCTCGCACGGSGTRTTGAACATC-3’) (Berge et al. 2014; Sarkar et al. 2004). 409-bp cts gene sequences were deposited in the GenBank database for seven isolates (accession no. OK105110-OK105116). Sequence of seven isolates shared 100% identity with several Pseudomonas cichorii strains within the GenBank database (accession no. KY940268 and KY940271), and the phylogenetic tree of cts genes of the seven isolates clustered with the phylogroup 11 of Pseudomonas syringae (accession no. KJ877799 and KJ878111), which was classified as P.cichorii. To satisfy Koch’s postulates, a pathogenicity test was tested by using a needle to dip a suspension of the bacterium (108 CFU/ml) and pricking three holes in the tobacco leaf. The control plants leaves were needled with sterile water. Each tobacco plant was inoculated with three leaves, and the test was repeated three times. All plants were placed in transparent plastic boxes and incubated in a greenhouse at 25 ± 3°C. The water-soaked spots appeared 24h after inoculation and quickly expanded through leaf veins. Three days after inoculation, all the inoculated leaves showed symptoms similar to those observed in the field. Control plants remained healthy. Only P. cichorii was successfully re-isolated from the lesions, confirming Koch’s postulates. Pseudomonas cichorii can infect eggplant, lettuce, tomatoand other crops, and has a wide range of hosts (Timilsina et al. 2017; Ullah et al. 2015). To our knowledge, this is the first report of P. cichorii causing leaf spot on tobacco in China.

Michelia alba (common name: white champaca), native to Indonesia, is a preciously ornamental and medicinal plant in the west and southeast of China and widely distributed in Nanning, Guangxi, China (Hou et al. 2018). In May 2020, a foliar disease of M. alba was observed in Nanning (22°51′ N; 108°17′ E), Guangxi, China, present on ca. 20-30% of the leaves. The disease began to develop from the margins of leaves in most cases. The symptoms recorded were light yellow spots, which gradually developed into ellipsoidal to irregular brown spots, surrounded by a wide yellow halo. The spots gradually enlarged in size and became grey-brown, with the dimension of 3.5 × 2.8 to 11.0 × 3.5 cm, even more than half of leaf area. In the later stage of infection, these spots coalesced resulting in necrosis and early shedding of the leaves. Sometimes black acervuli were observed on some lesions. For isolation of the fungus, ten symptomatic leaves were randomly sampled from five trees and washed with sterile water. Small pieces of infected tissue (about 4 mm2) were surface disinfected in 75% alcohol for 30 s and in 0.1% aqueous solution of mercury chloride for 1 min. Finally these tissue pieces were rinsed three times with sterile water, plated on potato dextrose agar (PDA) and then incubated for 7 days at 28℃ with a photoperiod of 12 h. Fifteen strains with similar morphological characterizations were isolated, and five representative isolates (BL-1 to BL-5) were purified. These cultures gave rise to grey-white colonies with bright orange conidial masses with contained one-celled, hyaline, guttulate conidia, measuring 12.68-20.70 × 4.27-7.84 µm (average 15.36 × 5.35 µm, n=100). Appressoria formed from conidia were brown, ellipsoidal or inverted trapezoid and measured 6.36-12.13 × 5.07-7.39 µm (average 8.29 × 6.36 µm, n=30). These morphological characteristics were similar to those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm identification, genomic DNA from mycelium of these five isolates was extracted, and the sequence of internal transcribed spacer (ITS), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), calmodulin (CAL) and β-tubulin (TUB2) were amplified (Zhang et al. 2020), and the GenBank accession numbers for the sequences were MW186173 to MW186177 (ITS), MW161290 to 161294 (CHS-1), MW161295 to MW161299 (GAPDH), MW161285 to 161289 (ACT), MW084710 to 084714 (CAL) and MW161300 to MW161304 (TUB2). The phylogenetic tree of six combined genes of the five isolates clustered with Colletotrichum siamense strains (CBS 125378, ICMP 17795 and ICMP 18121). Therefore, the isolates were identified as C. siamense. Five isolates (BL-1 to BL-5) were tested for pathogenicity. Wounded and unwounded detached healthy leaves were inoculated using mycelial discs (5 mm in diameter) and conidial suspensions (with the concentration of 1 × 105 conidia/ml) at the same time, incubated in a growth chamber at 25-30℃ (85-90% relative humidity, with a photoperiod of 12 h). Three leaves (wounded left half blade and unwounded right half blade) were inoculated with different methods for each isolate, and the tests were repeated three times. Four days after inoculation, leaf spots were observed on all wounded leaves, while 5-10% of the unwounded leaves showed lesions. Control leaves inoculated with PDA discs and sterile water remained symptomless. Colletotrichum. siamense was re-isolated from the lesions, confirming Koch's postulates. At least 60 plant species have been reported to be infected by C. siamense worldwide (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. alba in China.

Mesona chinensis is an important medicinal and edible plant resource distributed in eight provinces in southern China. In December 2021, an unknown stem and leaf blight disease was found in M. chinensis cultivation areas in Longzhou County, Guangxi, China. Sixty days after transplanting, the incidence of this disease was 10%. Leaf spots mostly appeared from the leaf edge, were irregular, brown to dark brown, causing more than half of the leaf or the whole leaf to die. The infected stem first showed dark brown spots, then constricted slightly, became necrotic and rotted with the expansion of the spots, resulting in the death of the whole plant. Loose cobweb-like mycelia, which resembled Rhizoctonia, could be seen on the diseased tissues in conditions of high humidity. To identify the pathogen, diseased stems and leaves with typical symptoms from Longzhou County were collected and surface-sterilized with 75% ethanol for 30 s. Small fragments (5×5 mm) at the junction of diseased and healthy tissues were disinfected with 1% NaClO for 1min, washed with sterile water three times, transferred to potato dextrose agar (PDA), and incubated at 28°C for 3 days. Mycelial tips were removed, and six isolates (No. R1-R6) were obtained. The colonies were initially gray white and later light brown. Many nearly round to irregular sclerotia appeared after 7 days of culture. The sclerotia turned from light brown to deep brown and were 1 to 5 mm in diameter. The mycelium branched at a 90° angle, with septa near the branches and a constriction of the mycelium at the base of the branch. These morphological characteristics were consistent with Rhizoctonia. For molecular identification, genomic DNA of the six isolates was obtained using an extraction kit (Biocolor, Shanghai, China), and primers ITS4/ITS5 were used to amplify the internal transcribed spacers (ITS) and 5.8S rRNA (White et al. 1990). A 750 bp DNA fragment was obtained and the sequences were deposited in GenBank (OM095383-OM095388). All isolates had ≥ 99% identity with anastomosis group AG1-1B (HG934429 and HQ185364) of R. solani. A phylogenetic tree showed that the isolates and those from anastomosis group AG1-1B clustered into one branch. To satisfy Koch’s postulates, the isolates from diseased leaf (No. R1, R2, and R3) and diseased stem (No. R4, R5, and R6) were inoculated on leaves and stems of 45-day-old M. chinensis plants. Five leaves and stems were inoculated with mycelial plugs of each isolate without wounding and another five leaves and stems were inoculated with mycelial plugs of each isolate after pinprick wounding. Control wounded leaves and stems were inoculated with sterile PDA discs. To maintain high humidity, the plants were incubated at 28°C and covered with transparent plastic covers. Diseased spots first appeared 24 h after inoculation. Three days post-inoculation, all inoculated leaves and stems showed symptoms like those observed in the field, whereas controls were asymptomatic. The pathogen was re-isolated from the diseased inoculated tissues using the method described above, and isolated fungi had the morphological characteristics of R. solani. Thus, the pathogen causing stem and leaf blight disease of M. chinensis was determined to be R. solani. The host range of R. solani is wide, and anastomosis group AG1-1B has been reported to infect plants such as rice, bean, fig, cabbage, and lettuce (Sneh et al. 1991). To our knowledge, this is the first report of R. solani causing a stem and leaf blight on M. chinensis, and provides a basis for diagnosis and control of the disease.