First Report of Bronzing Disease Caused by Pantoea stewartii on Jackfruit in China.

Jackfruit (Artocarpus heterophyllus) is an important tropical commercial fruit crop grown in Hainan province, China. In recent years, severe jackfruit bronzing disease has been found in 11 cities and counties in Hainan. On average, 80% of trees in a jackfruit orchard are affected once bronzing disease is detected. The disease is characterized by yellow-orange to reddish discoloration of the pulp and rags of infected fruit (Hernández-Morales et al. 2017). Jackfruit bronzing disease has been reported previously in the Philippines (Gapasin et al. 2012), Malaysia (Zulperi et al. 2017), and Mexico (Hernández-Morales et al. 2017). Diseased samples of jackfruit 'Tai Eight' with the bronzing symptoms were collected from a plantation in Changjiang, Hainan. The samples were sterilized with 75% ethanol for 30 s, then soaked with 1% sodium hypochlorite for 8 min, and rinsed with sterilized distilled water. The sterilized tissues were ground in 2 mL sterile water, and allowed to stand for 30 min. Then, 500 µL of the supernatant was spread on Glucose-Yeast agar medium and incubated overnight at 28ºC. Representative bacterial colonies were lemon-yellow, convex and smooth, transparent with entire edges. Colonies were Gram-negative, positive for catalase and gelatin liquefaction, which were consistent with the characteristics of P. stewartii subsp. stewartii. In PCR amplifications, an 920 bp amplicon of strain JTPE2 with the primers ES16/ESIG2c (Coplin et al. 2002) and an 1100 bp amplicon of strain JTPC2 with the primers CPSL1/CPSR2c (Ibrahim et al. 2019) were obtained, whereas no bands were observed for the negative control samples. The ES16/ESIG2c and CPSL1/CPSR2c fragments were sequenced for nucleotide BLAST (BLASTn) searches of the NCBI database and phylogenetic tree construction. The obtained ES16/ESIG2c sequences (SAR accession no. SRR22405292) showed 99.07%-99.60% similarity with P. stewartii subsp. stewartii (CP017581, AJ311838 and MF598163). The obtained CPSL1/CPSR2c sequences (SAR accession no. SRR22405293) showed 99.40%-99.99% similarity with P. stewartii subsp. stewartii (MW971422, MH752485 and MH257287). Phylogenetic analysis based on cpsDE sequences (Ibrahim et al. 2019) using the maximum likelihood method revealed that strains JTPE2 and JTPC2 were clustered together with P. stewartii subsp. stewartii. A pathogenicity test was conducted by injecting 2 mL of 108 CFU/ml bacterial suspension into pulp from healthy, surface-sterilized jackfruit. Pulp injected with sterilized distilled water served as a negative control. All inoculated samples produced bronzing symptoms from 2-3 weeks post-inoculation similar to the field-observed symptoms, whereas control fruit were asymptomatic. The strains were reisolated from symptomatic jackfruit pulp to complete Koch's postulates. The bacterial suspension was inoculated on 2-week-old maize seedlings to supplement in vivo pathogenicity testing. Typical Stewart's disease leaf symptoms were visible at 2 weeks post-inoculation. Based on morphological, biochemical, and physiological evidence, pathogenicity tests, and molecular analyses, the pathogenic bacterium isolated from 'Tai Eight' jackfruit was identified as P. stewartii subsp. stewartii. To our knowledge, this is the first report of bronzing disease caused by P. stewartii subsp. stewartii on jackfruit in China, which may assist in preventing the global spread of jackfruit bronzing disease.

First Report of Bark Split Disease Caused by Pectobacterium carotovorum on Artocarpus heterophyllus (Jackfruit) in China.

Jackfruit (Artocarpus heterophyllus) is widely cultivated in the tropical areas in the world. Jackfruit bark split disease occurred in the large-scale plantations of 18 cities and counties surveyed in Hainan since 2021, among which the incidence rate of serious orchards reached about 70%, and the mortality rate reached about 35%. Jackfruit bark split disease mainly harms tree branches and trunks, manifested as water stains, bark gumming, bark depression, bark cracking, and ultimately plant death. To identify the pathogen, Four samples with jackfruit bark split disease symptoms were collected, sterilized with 75% ethanol for 30 s, then soaked in 2% sodium hypochlorite (NaClO) for 5 mins, and finally rinsed continuously with sterilized distilled water. The sterilized tissues were placed on LB agar medium and incubated in illumination incubator at 28 ℃. Four milky white, round with neat edges, convex and smooth, translucent colonies were obtained. All isolates (JLPs-1 to JLPs-4) were Gram-negative, negative for oxidase, catalase and gelatin liquefaction. Amplification and sequencing of 16S rDNA gene from 4 isolates were conducted with the universal primers 27f /1492r (Lane et al. 1991). The BLASTn analysis of obtained JLPs-1 and JLPs-3 sequences (GenBank accession nos. OP942452 and OP942453) showed an identity percentage of 98.99% and 98.93% with Pectobacterium sp. (CP104733), respectively. Phylogenetic analysis based on 16S rDNA gene using the neighbor-joining method with MEGA 7.0 software revealed that JLPs-1 and JLPs-3 were clustered together with P. carotovorum reference strains. The four housekeeping genes gyrA, recA, rpoA and rpoS were partially sequenced for JLPs-1 isolates using primers gyrA1/gyrA4, recA1/recA2c, rpoS1/rpoS2 and rpoA F1/rpoA R1 (Loc et al. 2022), respectively. Multilocus sequence analyses identified the isolates from jackfruit as P. carotovorum. To further confirm the identification of Pectobacterium carotovorum, pelY gene, P. carotovorum subsp. Brasiliensis 16S-23S intergenic region (Pcb IGS) and P. carotovorum subsp. carotovorum (Pcc) specific fragment were amplified with primers Y1/Y2 (Darrasse et al. 1994), BR1f/L1r (Duarte et al. 2004) and EXPCCF/EXPCCR (Kang et al. 2003), respectively. A 540 bp target fragment was successfully amplified from JTPs only by EXPCCF/EXPCCR and there no bands for the other two primers. Pathogenicity test was performed in the field, and all the inoculated trees were 2-3-year-old 'Qiong Yin No.1' variety. Dense small holes were pierced with sterilized inoculation needle on four healthy jackfruit trees. Then punctured wounds were spraying-inoculated with bacteria suspension of JLPs-1 (108 CFU/ml), and finally wrapped with plastic wrap to moisturize. Two trees inoculated with sterile distilled water served as negative control. Typical symptoms of bark gumming, bark depression, bark cracking were observed on all of the inoculated trees at 17 dpi which just similar to those originally caused by P. carotovorum in the field, whereas negative control trees remained asymptomatic. The strains were re-isolated successfully from symptomatic jackfruit trees and were consistent with the biological and molecular biological characteristics of original strains, confirming that the pathogen of jackfruit bark split disease was Pectobacterium carotovorum. To our knowledge, this is the first report of P. carotovorum causing bark split disease on jackfruit in China.

Genome-wide identification and expression analysis of carotenoid cleavage oxygenase genes in Litchi (Litchi chinensis Sonn.).

BACKGROUND: Carotenoid cleavage oxygenases (CCOs) include the carotenoid cleavage dioxygenase (CCD) and 9-cis-epoxycarotenoid (NCED), which can catalize carotenoid to form various apocarotenoids and their derivatives, has been found that play important role in the plant world. But little information of CCO gene family has been reported in litchi (Litchi chinensis Sonn.) till date. RESULTS: In this study, a total of 15 LcCCO genes in litchi were identified based on genome wide lever. Phylogeny analysis showed that LcCCO genes could be classified into six subfamilies (CCD1, CCD4, CCD7, CCD8, CCD-like, and NCED), which gene structure, domain and motifs exhibited similar distribution patterns in the same subfamilies. MiRNA target site prediction found that there were 32 miRNA target sites in 13 (86.7%) LcCCO genes. Cis-elements analysis showed that the largest groups of elements were light response related, following was plant hormones, stress and plant development related. Expression pattern analysis revealed that LcCCD4, LcNCED1, and LcNCED2 might be involving with peel coloration, LcCCDlike-b might be an important factor deciding fruit flavor, LcNCED2 and LcNCED3 might be related to flower control, LcNCED1 and LcNCED2 might function in fruitlet abscission, LcCCD4a1, LcCCD4a2, LcCCD1, LcCCD4, LcNCED1, and LcNCED2 might participate in postharvest storage of litchi. CONCLUSION: Herein, Genome-wide analysis of the LcCCO genes was conducted in litchi to investigate their structure features and potential functions. These valuable and expectable information of LcCCO genes supplying in this study will offer further more possibility to promote quality improvement and breeding of litchi and further function investigation of this gene family in plant.

Genome-wide characterization and expression profiling of B3 superfamily during ethylene-induced flowering in pineapple (Ananas comosus L.).

BACKGROUND: The B3 superfamily (B3s) represents a class of large plant-specific transcription factors, which play diverse roles in plant growth and development process including flowering induction. However, identification and functional surveys of B3 superfamily have not been reported in ethylene-induced pineapple flowering (Ananas comosus). RESULTS: 57 B3 genes containing B3 domain were identified and phylogenetically classified into five subfamilies. Chromosomal localization analysis revealed that 54 of 57 AcB3s were located on 21 Linkage Groups (LG). Collinearity analysis demonstrated that the segmental duplication was the main event in the evolution of B3 gene superfamily, and most of them were under purifying selection. The analysis of cis-element composition suggested that most of these genes may have function in response to abscisic acid, ethylene, MeJA, light, and abiotic stress. qRT-PCR analysis of 40 AcB3s containing ethylene responsive elements exhibited that the expression levels of 35 genes were up-regulated within 1 d after ethephon treatment and some were highly expressed in flower bud differentiation period in stem apex, such as Aco012003, Aco019552 and Aco014401. CONCLUSION: This study provides a basic information of AcB3s and clues for involvement of some AcB3s in ethylene-induced flowering in pineapple.

The UDP glucose: flavonoid-3-O-glucosyltransferase (UFGT) gene regulates anthocyanin biosynthesis in litchi (Litchi chinesis Sonn.) during fruit coloration.

We analyzed a litchi cultivar that included three phenotypes for pericarp color, ranging from green, indicating the absence of anthocyanins, to yellow, and red. Anthocyanins, chlorophylls, carotenoids, and flavonoids were measured in the three stages. Fruit coloration of red-skinned litchi was mainly due to higher flavonols, and anthocyanin pigments, lower chlorophyll (higher chlorophyll degradation). Expression of four genes of the anthocyanin pathway coding for phenylalanine ammonialyase, chalcone synthase, flavanone-3-hydroxylase, and the UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT), was analyzed by RT-PCR at three developmental stages from before the onset of ripening to full maturity. Gene expression patterns were compared to anthocyanin metabolites. The contents of anthocyanins and flavonols in the pericarps were consistent with the higher mRNA levels of UFGT, while, transcription of the other gene was not expected to follow the anthocyanin content. We suggest that UFGT might play an important role in anthocyanin biosynthesis in the pericarp of litchi. Thus, UFGT expression strongly influences fruit coloration in litchi.

Identification and verification of key taste components in wampee using widely targeted metabolomics.

Due to the lack of comprehensive evaluation of all metabolites in wampee, the metabolic reasons for taste differences are unclear. Here, two local varieties YF1 (sweet taste) and YF2 (sweet-sour taste), were selected for quality analysis, followed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based widely targeted metabolomic analysis. YF1 and YF2 were clearly separated by principal component analysis (PCA) and cluster analysis, and 449 metabolites were different between the cultivars, including 29 carbohydrates and 29 organic acids. Among them, d-galactose, d-mannose, and d-fructose 6-phosphate contributed mainly to the sweet taste of the YF1 wampee. l-citramalic acid, 2-hydroxyglutaric acid, and 3-methylmalic acid were the dominant organic acids in YF2 wampee, and therefore, contributed primarily to the sweet-sour taste. The differential metabolites were significantly enriched in the "ascorbate and aldarate metabolism" and "C5-branched dibasic acid metabolism" pathways. Ascorbate played a crucial role in the regulation of sugars and organic acids through those pathways. In addition, high-performance liquid chromatography (HPLC) based quantitative verification exhibited the same specific cultivar variations.

Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn.

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m(-2) among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red ('Kuixingqingpitian', 'Xingqiumili', 'Yamulong'and 'Yongxing No. 2'), unevenly red ('Feizixiao' and 'Sanyuehong') and fully red ('Meiguili', 'Baila', Baitangying' 'Guiwei', 'Nuomici' and 'Guinuo'). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.