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.

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.

Metabolic Profiling and Potential Taste Biomarkers of Two Rambutans during Maturation.

The metabolite-caused taste variation during rambutan maturation is unknown due to a lack of systematic investigation of all components. In this study, three growing stages, including unripe (S1), half-ripe (S2), and full-ripe (S3) BY2 and BY7 rambutans were compared and profiled by UPLC-MS/MS-based widely targeted metabolomics analysis. We demonstrated that the sugar-acid ratios of two rambutans were greatly improved between the S2 and S3 stages. A total of 821 metabolites were identified, including 232, 205, 204, and 12 differential metabolites (DMs) in BY2-S1 vs. BY2-S2, BY2-S2 vs. BY2-S3, BY7-S1 vs. BY7-S2, and BY7-S2 vs. BY7-S3, respectively. A correlation analysis showed that gamma-aminobutyric acid (GABA) could be the sugar-acid ratio biomarker of BY2 rambutan. Methionine (Met), alanine (Ala), and S-methyl-L-cysteine (SMC) could be total amino acid biomarkers of BY2 and BY7 rambutans. In addition, UPLC-MS/MS-based quantitative verification of the above biomarkers exhibited the same variations as metabolomics analysis. This study not only provides useful nutritive information on rambutans but also valuable metabolic data for rambutan breeding strategies.

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.

Intercropping Pinto Peanut in Litchi Orchard Effectively Improved Soil Available Potassium Content, Optimized Soil Bacterial Community Structure, and Advanced Bacterial Community Diversity.

Intercropping is widely used in agricultural production due to its capability of raising land productivity and providing an opportunity to achieve sustainable intensification of agriculture. In this study, soil samples from 10 to 20 cm depth of intercropping Pinto peanut in litchi orchard and litchi monoculture mode were established to determine soil attributes, enzyme activities, as well as the effect on soil bacterial diversity. On this basis, 16S rRNA V4-V5 region of soil bacterial communities in litchi/Pinto peanut intercropping (LP) mode and litchi monoculture mode (CK) was detected by the Illumina MiSeq sequencing platform. The results showed that the content of available potassium (AK) in LP was significantly higher than that in CK by 138.9%, and the content of available nitrogen (AN) in LP was significantly lower than that in CK by 19.6%. The soil enzyme activities were higher in LP as a whole, especially sucrase (SC) and acid protease (PT) were significantly higher by 154.4 and 76.5%, respectively. The absolute abundance and alpha diversity of soil microbiota were significantly higher in the intercropping group. Most importantly, endemic species with a significant difference in LP was higher by ~60 times compared to CK treatment. In the aspect of soil bacterial community structure, the dominant phyla of the two groups were Acidobacteria, Proteobacteria, Chloroflexi, and Actinobacteria. At the genus level, the absolute abundance of Flavobacterium and Nitrososphaera was significantly higher by 79.20 and 72.93%, respectively, while that of Candidatus_Koribacter was significantly lower with an amplitude of 62.24% in LP than in CK. Furthermore, the redundancy analysis (RDA) suggested that AK, which was highly associated with the dominant genera and phyla, is the vitally dominating environmental factors in LP groups, while in CK groups, it is AN and pH. In addition, PICRUSt2 analysis indicated that intercropping improved the metabolic activity of bacteria which can be correlated to the resistance of litchi root systems to soil-borne diseases. Overall, this study is expected to provide a theoretical basis and technical support for the healthy intercropping cultivation of 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.

Development of molecular markers based on the promoter difference of LcFT1 to discriminate easy- and difficult-flowering litchi germplasm resources and its application in crossbreeding.

BACKGROUND: Litchi is a well-known subtropical fruit crop. However, irregular bearing attributed to unstable flowering is a major ongoing problem for the development of the litchi industry. In a previous study, our laboratory proved that litchi flowering was induced by low temperature and that a FLOWERING LOCUS T (FT) homologue gene named LcFT1 played a pivotal role in this process. The present study aimed to understand the natural variation in FT among litchi germplasm resources and designed markers to verify easy- and difficult-flowering litchi germplasms. A grafting experiment was also carried out to explore whether it could shorten the seedling stage of litchi seedlings. RESULTS: Two types of LcFT1 promoter existed in different litchi germplasm resources, and we named them the 'easy-flowering type of LcFT1 promoter' and 'difficult-flowering type of LcFT1 promoter', which resulted in three different LcFT1 genotypes of litchi germplasm resources, including the homozygous easy-flowering type of the LcFT1 genotype, homozygous difficult-flowering type of the LcFT1 genotype and heterozygous LcFT1 genotype of litchi germplasm resources. The homozygous easy-flowering type of the LcFT1 genotype and heterozygous LcFT1 genotype of the litchi germplasm resources completed their floral induction more easily than the homozygous difficult-flowering type of the LcFT1 genotype of litchi germplasm resources. Herein, we designed two kinds of efficient molecular markers based on the difference in LcFT1 promoter sequences and applied them to identify of the easy- and difficult-flowering litchi germplasm resources. These two kinds of molecular markers were capable of clearly distinguishing the easy- from difficult-flowering litchi germplasm resources at the seedling stage and provided the same results. Meanwhile, grafting the scion of seedlings to the annual branches of adult litchi trees could significantly shorten the seedling stage. CONCLUSIONS: Understanding the flowering characteristics of litchi germplasm resources is essential for easy-flowering litchi breeding. In the present study, molecular markers provide a rapid and accurate approach for identifying the flowering characteristics. The application of these molecular markers not only significantly shortened the artificial crossbreeding cycle of easy-flowering litchi cultivars but also greatly saved manpower, material resources and land.

Identification of Genes Involved in Low Temperature-Induced Senescence of Panicle Leaf in Litchi chinensis.

Warm winters and hot springs may promote panicle leaf growing and repress floral development. To identify genes potentially involved in litchi panicle leaf senescence, eight RNA-sequencing (RNA-Seq) libraries of the senescing panicle leaves under low temperature (LT) conditions and the developing panicle leaves under high temperature (HT) conditions were constructed. For each library, 4.78⁻8.99 × 106 clean reads were generated. Digital expression of the genes was compared between the senescing and developing panicle leaves. A total of 6477 upregulated differentially expressed genes (DEGs) (from developing leaves to senescing leaves), and 6318 downregulated DEGs were identified, 158 abscisic acid (ABA)-, 68 ethylene-, 107 indole-3-acetic acid (IAA)-, 27 gibberellic acid (GA)-, 68 cytokinin (CTK)-, 37 salicylic acid (SA)-, and 23 brassinolide (BR)-related DEGs. Confirmation of the RNA-Seq data by quantitative real-time PCR (qRT-PCR) analysis suggested that expression trends of the 10 candidate genes using qRT-PCR were similar to those revealed by RNA-Seq, and a significantly positive correlation between the obtained data from qRT-PCR and RNA-Seq were found, indicating the reliability of our RNA-Seq data. The present studies provided potential genes for the future molecular breeding of new cultivars that can induce panicle leaf senescence and reduce floral abortion under warm climates.

Differential gene expression between the vigorous and dwarf litchi cultivars based on RNA-Seq transcriptome analysis.

Litchi (Litchi chinesis Sonn.) is the most economically significant member of Sapindaceae family, especially in sub-tropical regions. However, its tall tree body often brings many inconveniences to production management. In order to modify the tree size or growth for productivity optimization and simplifying management, it is urgent to reveal the dwarf mechanism of litchi for dwarfing rootstocks or cultivar breeding. However, to date, the mechanisms on litchi dwarfism is still poor known. In the present study, transcriptome profiling were performed on L. chinensis cv. 'Feizixiao' (FZX, vigorous cultivar) and 'Ziniangxi' (ZNX, dwarf cultivar). A total of 55,810 unigenes were obtained, and 9,190 unigenes were differentially expressed between vigorous and dwarf litchi samples. Gene functional enrichment analysis indicated that the differentially expressed unigenes (DEGs) were related to phytohormone metabolism and signal transduction, and energy metabolism pathways. In particular, GA2ox were only up-regulated in ZNX samples, indicating GA might play an important role in regulating huge difference between vigorous and dwarf litchi cultivars. In addition, the 35S::LcGA2ox transgenic tobacco plants were dwarf and had smaller leaves or branches than wild type plants. Our study provided a series of candidate genes to reveal the mechanism of litchi dwarf.

Aberrant seed development in Litchi chinensis is associated with the impaired expression of cell wall invertase genes.

Cell wall invertase (CWIN) are known to play important roles in seed development. However, most reports to date have focused on a single gene family member, and have mainly investigated CWIN functions during the filling stage of seed development. In this study, we found significant lower levels of CWIN protein and activity associated with seed abortion in the Litchi chinensis cultivar "Nuomici." We identified five litchi CWIN genes and observed that the expression of LcCWIN5 was limited to the flower tissues and decreased sharply with fruit development. Silencing of LcCWIN5 expression before 28 DAA (cell division stage) resulted in perturbed liquid endosperm development, smaller seeds, and higher seed abortion rate, while silencing after 28 DAA (filling stage) had no effect on seed development. In contrast, LcCWIN2 was mostly expressed in the funicle and seed coat, and increased with fruit development. Decreased LcCWIN2 expression and CWIN activity during early seed filling coincided with smaller seeds in the cultivar "Feizixiao." Silencing of LcCWIN2 caused a reduction in the seed size without inducing seed abortion. We propose that CWIN activity in seed maternal tissues during cell division stage is likely due to LcCWIN5 expression, which regulates early seed development. On the other hand, CWIN activity during the filling stage is due to the expression of LcCWIN2, which may promote carbon import by creating a sucrose gradient. Comparable LcCWIN5 expression, but much lower CWIN activity, detected in the funicle of "Nuomici" is consistent with post-translational regulation.

Transcriptome changes between compatible and incompatible graft combination of Litchi chinensis by digital gene expression profile.

Plant grafting has been practiced widely in horticulture and proved as a useful tool in science. However, the mechanisms of graft healing or graft incompatibility remain poorly understood. In this study, Litchi chinensis cv. 'Jingganghongnuo' homograft ('J/J') and 'Jingganghongnuo'/'zhuangyuanhong' heterograft ('J/Z') as compatible and incompatible combination, respectively, was used to study transcriptional changes between incompatible and compatible graft during graft union formation. Anatomical observation indicated that three stages (2 h, 14 d and 21 d after grafting) were critical for graft union formation and selected for high-throughput sequencing. Results indicated 6060 DEGs were differentially expressed in the compatible combination and 5267 DEGs exhibiting in the incompatible one. KEGG pathway enrichment analysis revealed that DEGs were involved in metabolism, wound response, phenylpropanoid biosynthesis and plant hormone signal transduction. The expression of 9 DEGs annotated in auxin pathway was up-regulated in compatible combination than that in incompatible combination. The IAA concentration confirmed that the IAA might promote the graft compatibility. In addition, 13 DEGs related to lignin biosynthesis were differentially expressed during graft healing process. Overall, our results provide abundant sequence resources for studying mechanisms underlying graft compatibility and establish a platform for further studies of litchi and other evergreen fruit trees.

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.