First report of Xanthomonas albilineans causing leaf scald on two chewing cane clones in Zhejiang province, China.

In recent years, outbreaks of leaf scald have been reported in two chewing cane clones "Guangdong Huangpi" and "Taoshang Guozhe" in Zhejiang province, China. From May to July 2019, we collected 11 and 13 leaf or stalk samples from symptomatic "Guangdong Huangpi" from four farms in Wenling and "Taoshang Guozhe" clones from three farms in Ruian, Zhejiang province, respectively. Leaves in young plants exhibited white pencil-line streaks (Supplement Fig. 1A & 1D) as well as partial or complete chlorosis of the leaf blade (Supplement Fig. 1B & 1E). Internal symptoms included an orange-red discoloration of the vascular bundles at the basal nodes of the stalk and discoloration extension into the internodes (Supplement Fig. 1C & 1F). Leaf and stalk tissues were used for bacterial isolation and purification on XAS medium, which is selective for Xanthomonas albilineans (Davis et al. 1994), using the streak plate method to obtain 24 isolates (Lin et al. 2018). Circular, convex, smooth, shiny and yellow colonies were isolated from all the samples. The pathogenicity of two isolates, XaCN30 from "Guangdong Huangpi" and XaCN43 from "Taoshang Guozhe", was confirmed with Koch's postulates according to the protocol reported by Lin et al. (2018). The incidences of diseased plants (56% and 63%) were observed in individual host clones at 28 d post-inoculation with isolates XaCN30 and XaCN43, respectively. Furthermore, all isolates were confirmed as X. albilineans via molecular methods. PCR amplification was conducted for all 24 isolates using the primer pairs XgyrB1F/XgyrB1R2 (Ntambo et al. 2019) and XAF1/XAR1 (Wang et al. 1999), which targeting the gyrB (encoding the b subunit of the DNA gyrase) and abc (encoding an ABC transporter) genes, and generating 904 bp and 608 bp amplicons, respectively. The PCR fragments were cloned into the pMD19-T vector (TaKaRa, Dalian, China). For each isolate, three single colonies of transformed Escherichia coli DH5α carrying targeted fragment were sequenced. These sequences were deposited into the GenBank with accession no. MT776053-MT776059 and MT776061-MT776077 for gyrB gene and MT776098-MT776104 and MT776106-MT776122 for abc gene. Based on the two concatenated DNA sequences of our 24 isolates, compared with 27 previously reported X. albilineans isolates obtained from the GenBank database, pairwise sequence identity analysis revealed that all 24 isolates from Zhejiang province had 99.4-100% identity with each other, 99.6-100% identity with 14 published domestic isolates, and 98.3-100% identity with 13 foreign isolates. Furthermore, phylogenetic analysis with MEGA 7.0 (Kumar et al. 2016) showed that the isolates from Zhejiang province clustered into two distinct groups (Supplement Fig. 2). One group consisted of 25 Chinese isolates (including all 11 isolates from Wenling) along with four isolates from the French West Indies (GPE PC73, GPE PC17, GPE PC86, and MTQ032), and one isolate from the USA (XaFL07-1), which were assigned to pulsed-field gel electrophoresis (PFGE) group B (Davis et al. 1997; Pieretti et al. 2012). A putative group was also proposed, which included all 13 isolates from Ruian, indicating that isolates from Ruian are distinct from the isolates isolated from other Chinese sugarcane-planting areas, including Wenling. We conclude that leaf scald disease in local clones of chewing cane are caused by X. albilineans in Zhejiang province in China, which will be helpful for leaf scald management in chewing cane, a cash crop.

First Report of Pantoea stewartii subsp. stewartii Causing Bacterial Leaf Wilt of Sugarcane in China.

The pathogen of Pantoea stewartii subsp. stewartii (Pss) that is the causal agent of Stewart's bacterial wilt of corn also infects numerous experimental hosts of graminaceous plants (Pepper et al., 1967; Wang et al., 2012). However, little is known about this pathogen naturally infecting sugarcane. In 2017, we observed some sugarcane cultivars showing leaf blade bleaching at the disease initiation stage, which further resulted in development of blight and necrotic lesions (Figure 1-A and -B) in Zhanjiang, Guangdong province of China. To diagnose this putative disease, five symptomatic leaf samples were collected from different sugarcane cultivars. The Pss was found to infect these samples using the nested PCR with Pss-specific outer primers PS1/PS4 and inner primers Ps2r/Ps3r that targeted at the 16S rRNA gene of this pathogen (Wang et al., 2009). The expected 262-bp fragments from positive samples were amplified, cloned, and sequenced (GenBank accession no. MW015795-MW015799). BLASTN analysis revealed that these isolates had more than 99.5% nucleotide identify (222 bp out of 262 bp) with each other and with Pss strains (ATCC 8199 and DC283) as well as P. stewartii subsp. indologenes strains (SR2-12 and LMG 2632) after sequences were trimmed at the 5'- and 3'-terminal of inner primer sequences. In addition, these leaf samples were surface-sterilized with 75% alcohol followed by macerated and chopped in sterile water. Upon plating on solid NA medium at 28 °C for 24-36 h, the bacterial colonies exhibited yellow color with circular, convex, smooth and translucent edges (Figure 1-C). Straight rods and non-encapsulated cells were detected under transmission electron microscopy (Figure 1-D). Moreover, an identical colony termed as PSCN1 was isolated from sugarcane cultivar YZ08-1095 and was further confirmed by the PCR with a universal primer pair 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1387R (5'-GGGCGGWGTGTACAAGGC-3') that targeted at bacterial 16S rRNA gene (Marchesi et al., 1998). A 1362-bp DNA fragment sequence was obtained from PSCN1 strain and deposited on GenBank library (accession no. MW015767). Sequence analysis showed that PSCN1 shared 99.9-100% nucleotide identity (1315 bp out of 1362 bp) with the two reference strains of Pss (ATCC 8199 and DC283) after sequences were trimmed at the 5'- and 3'-terminal of primer sequences. According to Koch's postulates, pathogenicity test was carried out on YZ08-1095 plants with 3-5 fully developed leaf inoculated with the suspended cells (108 cells/ml) of PSCN1 strain by cutting the one-third of leaves before spraying with a suspension. Control plants were mock-inoculated with serial liquid nutrition agar medium. Two independent experiments were performed for pathogenicity assay and more than 28 plants of YZ08-1095 were used in each treatment. Plants were cultured in a growth chamber at 28 °C and 60% humidity under a 16 h light/8 h dark photoperiod. Leaves inoculated by the PSCN1 initially showed bleached, blight and wilting symptoms on leaf edges at seven days post-inoculation (dpi) (Figure 1-E and -F), which were similar to those symptoms observed in the fields. Control plants remained asymptomatic (Figure 1-G). The average incidence of diseased plant was 51.9% at 21 dpi. The bacteria were subsequently re-isolated from diseased leaves, and yielded colonies were completely identical to the PSCN1. Taken together, our data provides the valuable information for diagnosis and controlling this disease in sugarcane.

[Trait stability and test site representativeness of sugarcane varieties based on GGE-biplot analysis].

Arithmetic mean method is commonly used to evaluate the yield stability and adaptability of sugarcane varieties, and variance analysis is applied to estimate the errors in regional trials. However, it is difficult to accurately evaluate the differences of the varieties due to the discrepancies across test sites and years. In this paper, GGE-biplot method was adopted to analyze the data from the regional trials with seven sugarcane varieties at five sites from 2008 to 2009, aimed to objectively evaluate the yield stability and adaptability of sugarcane varieties in China. Among the test sugarcane varieties, Funong No. 30 had higher cane yield and better yield stability, Yuegan No. 18 had higher sugar content and better trait stability, Funong No. 28 and Yunzhe 99-91 had high sucrose content and trait stability, while Yuegan No. 16 had the highest cane yield and sugar content but ordinary stability. In the test sites, Zhangzhou City in Fujian Province and Suixi City in Guangdong Province had the best representativeness and discrimination. This study showed that GGE-biplot analysis provided a simple and effective method to analyze the high yield and stability of sugarcane varieties in regional trials, and supplied the basis for the approval and extension of new sugarcane varieties.