Comparative Transcriptome and sRNAome Analysis Suggest Coordinated Citrus Immune Responses against Huanglongbing Disease.

Citrus Huanglongbing (HLB), caused by the phloem-inhibiting bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease, intimidating citrus production worldwide. Although commercially cultivated citrus cultivars are vulnerable to CLas infection, HLB-tolerant attributes have, however, been observed in certain citrus varieties, suggesting a possible pathway for identifying innate defense regulators that mitigate HLB. By adopting transcriptome and small RNAome analysis, the current study compares the responses of HLB-tolerant lemon (Citrus limon L.) with HLB-susceptible Shatangju mandarin (Citrus reticulata Blanco cv. Shatangju) against CLas infection. Transcriptome analysis revealed significant differences in gene expression between lemon and Shatangju. A total of 1751 and 3076 significantly differentially expressed genes were identified in Shatangju and lemon, respectively. Specifically, CLas infected lemon tissues demonstrated higher expressions of genes involved in antioxidant enzyme activity, protein phosphorylation, carbohydrate, cell wall, and lipid metabolism than Shatangju. Wet-lab experiments further validated these findings, demonstrating increased antioxidant enzyme activity in lemon: APX (35%), SOD (30%), and CAT (64%) than Shatangju. Conversely, Shatangju plants exhibited higher levels of oxidative stress markers like H2O2 (44.5%) and MDA content (65.2%), alongside pronounced ion leakage (11.85%), than lemon. Moreover, microscopic investigations revealed that CLas infected Shatangju phloem exhibits significantly more starch and callose accumulation than lemon. Furthermore, comparative sRNA profiles revealed the potential defensive regulators for HLB tolerance. In Shatangju, increased expression of csi-miR166 suppresses the expression of disease-resistant proteins, leading to inadequate defense against CLas. Conversely, reduced expression of csi-miR166 in lemon plants enables them to combat HLB by activating disease-resistance proteins. The above findings indicate that when infected with CLas, lemon exhibits stronger antioxidative activity and higher expression of disease-resistant genes, contributing to its enhanced tolerance to HLB. In contrast, Shatangju shows lower antioxidative activity, reduced expression of disease-resistant genes, significant ion leakage, and extensive callose deposition, possibly related to damage to plant cell structure and blockage of phloem sieve tubes, thereby promoting the development of HLB symptoms.

Carvacrol/ß-cyclodextrin inclusion complex as a fumigant to control decay caused by Penicillium digitatum on Shatangju mandarin slices.

Preservation and microorganism control of fresh-cut fruit pose a persistent challenge in the food industry. To address this issue, we prepared a ß-cyclodextrin (ß-CD) inclusion complex containing carvacrol using a coprecipitation method and employed it for the non-contact fumigation of fresh-cut Shatangju mandarin slices. This biodegradable and safe preservative offers an effective means to combat spoilage and ensure product quality. We confirmed the formation of the encapsulated structure of the inclusion complex through various characterization methods, including scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and differential thermal analysis (DTA). We also demonstrated the inhibitory effect of this preservative on Penicillium digitatum and its associated spoilage both in vitro and in vivo. The incidence and severity were significant lower in the inclusion complex-treated group (75.0% and 46.7%, respectively) compared to the group treated with pure carvacrol (100% and 69.2%, respectively). In addition, fruit freshness parameters and sensory evaluation showed that the inclusion complex treatment effectively maintained the overall quality of the fruit and achieved the highest consumer acceptance.

Small RNAs contribute to citrus Huanglongbing tolerance by manipulating methyl salicylate signaling and exogenous methyl salicylate primes citrus groves from emerging infection.

Citrus production is severely threatened by the devastating Huanglongbing (HLB) disease globally. By studying and analyzing the defensive behaviors of an HLB-tolerant citrus cultivar 'Shatangju', we discovered that citrus can sense Candidatus Liberibacter asiaticus (CLas) infection and induce immune responses against HLB, which can be further strengthened by both endogenously produced and exogenously applied methyl salicylate (MeSA). This immune circuit is turned on by an miR2977-SAMT (encoding a citrus Salicylate [SA] O-methyltransferase) cascade, by which CLas infection leads to more in planta MeSA production and aerial emission. We provided both transgenic and multi-year trail evidences that MeSA is an effective community immune signal. Ambient MeSA accumulation and foliage application can effectively induce defense gene expression and significantly boost citrus performance. We also found that miRNAs are battle fields between citrus and CLas, and about 30% of the differential gene expression upon CLas infection are regulated by miRNAs. Furthermore, CLas hijacks host key processes by manipulating key citrus miRNAs, and citrus employs miRNAs that coordinately regulate defense-related genes. Based on our results, we proposed that miRNAs and associated components are key targets for engineering or breeding resistant citrus varieties. We anticipate that MeSA-based management, either induced expression or external application, would be a promising tool for HLB control.

First report of Neopestalotiopsis rosae causing leaf blight on Shatangju in southern China.

Shatangju (Citrus reticulata Blanco cv. Shatangju) belongs to genus Citrus and was cultivated extensively in southern China. In April 2022, a leaf blight-like symptom (firstly brown spots appeared on infecting leaves, then these brown spots extended, finally the whole leaves displayed blight-like symptom) was observed on 5%~10% of Shatangju seedlings (around five hundreds in total) in an orchard located in Wuhan city, Hubei, China. Diseased leaves from three seedlings were collected and cut into pieces (0.2 to 0.5 cm). These pieces were surface-sterilized using 75% ethanol for 3 min, rinsed with sterile distilled water for several times, then placed on potato dextrose agar (PDA) and incubated at 26°C with 12-h light/dark cycle. Over 20 pieces plated, wherein 30% were identified as Colletotrichum fructicola, 60% as Neopestalotiopsis spp., and 10% developed saprophytes. C. fructicola was a known pathogen on citrus, thus Neopestalotiopsis spp. was further investigated. Eight single-conidium colonies of the Neopestalotiopsis spp. were obtained, wherein STJ-8 was chosen as a representative for further study. The average growth rate of STJ-8 was 15.1±0.5 mm/day (n=5). Fungal colonies produced white cottony mycelium with abundant black acervuli distributed in concentric rings 6-8 days after planting, which ranged from 342.3 to 710.5 µm in diameter (n=100). Conidia were fusoid, five cells, four septa with average dimensions of 25.36×5.47 µm (n=100). Basal and apical cells were hyaline, wherein three middle cells were brown with darker septa. The apical cell was cylindrical with two to three transparent accessory filaments (13.7 to 30.5 µm in length, n=80). Basal cell was conic with an appendage (4.1 to 8.8 µm in length, n=40). Partial sequences of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and ß-tubulin (TUB2) were amplified with reported primers (White et al. 1990; Lee et al. 2006; Maharachchikumbura et al. 2014), sequenced, and submitted to GenBank (accession nos. ITS: OP236541; TEF-1α: OP250124; TUB2:OP263094). BLASTn results showed 100% identity with the corresponding sequences of Neopestalotiopsis rosae. A multilocus phylogenetic analysis showed STJ-8 was closest to N. rosae. Thus, STJ-8 was identified as N. rosae. Pathogenicity tests were performed on one-year-old Shatangju seedlings and detached primary leaves by inoculating needle-wounded leaves with seven days old 5-mm mycelial plugs/acervuli (about 5000 spores) of STJ-8. Control seedlings/leaves were inoculated with 5-mm PDA plugs/sterile water drops. All inoculated detached leaves were cultured at same the place with STJ-8 cultured, while inoculated seedlings were put in a growth chamber at 26°C under a 16-h light/dark cycle (60% humidity). Symptoms developed on all inoculated leaves (except healthy control) 2 and 4 days post-inoculation by mycelial plugs and acervuli, respectively. N. rosae was re-isolated from the inoculated leaves, confirming Koch's postulates. N. rosae has been reported to cause diseases on various plants worldwide (Rebollar-Alviter et al. 2020; Xavier et al. 2021; Lawrence et al. 2022). In China, N. rosae has been reported to cause leaf spot/blight on pecan and strawberry (Wu et al. 2021; Gao et al. 2022), which caused great loss on these crops. To our knowledge, this is the first report of N. rosae causing leaf disease on citrus. Our study is important for developing control strategies against N. rosae in future.

Transcriptome analyses reveal the potential mechanisms for color changes of a sweet orange peel induced by Candidatus Liberibacter asiaticus.

'Shatangju' mandarin (Citrus reticulate Blanco cv. Shatangju) is a Chinese citrus specialty in southern China with a delicious taste and an attractive appearance. Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) threatens the Shatangju industry seriously. Fruits from citrus trees with HLB show 'red nose' peels with a serious reduction in fruit value. Differentially expressed genes (DEGs) have been identified in the leaves of several citrus species with HLB infection. However, similar studies on the fruit peels of citrus trees with HLB infection are very limited. In this study, the pathogen CLas was diagnosed in the 'red nose' fruit peels of Shatangju. The chlorophyll and carotenoid contents in different peels were also analyzed. Besides, we identified DEGs in the comparison between peels from normal red-colored and 'red nose' fruits via RNA-seq. A total of 1922 unigenes were identified as DEGs, of which 434 were up-regulated and 1488 were down-regulated in the 'red nose' fruit peels. DEGs involved in chlorophyll and carotenoids biosynthesis, photosynthesis, and transcription factors could be responsible for fruit color changes after HLB infection. Our findings provide a preliminary understanding of the mechanism underlying the formation of a 'red nose' on fruit peel from HLB-infected trees.

First Report of Curvularia lunata Causing Leaf Spot on Shatangju in China.

Shatangju (Citrus reticulata Blanco) is an economically important citrus cultivar in China. It has a unique flavor and is very popular among consumers. In May 2018, we observed leaf spots on ~20% of Shatangju trees in a 7 ha orchard in Huaiji country, Guangdong, China. Small maroon spots initially developed on the lower leaf surfaces of the symptomatic trees, which then expanded and coalesced into larger lesions. The lesions became visible from the upper leaves, with light gray centers and dark brown margins surrounded by yellow halos. The infected leaves would finally become wilted. To isolate the pathogen, we cut 0.5 × 0.5 cm2 squares from the lesion margins of the symptomatic leaves, disinfested them using 1% NaClO for 20 s, and then with 70% ethanol for 1 min, rinsed them in sterile distilled water three times, and inoculated them onto three potato dextrose agar (PDA) plates (5 squares/plate), which were kept under 25 °C in the dark. Colonies with similar appearance were observed on all the plates and subcultured via the single-spore method (Ho et al. 1997). The mycelia of the subcultures gradually turned from white to black. The colonies were morphologically close to the fungal species Curvularia lunata (Wakker) Boedijn (Ellis 1971). At 8 days after inoculation, the conidia were 18.9-25.1 µm × 8.1-11.6 µm in size (n=50), fusiform or geniculate, smooth-walled, dark-brown, 3-septate, and with a slightly curved second cell and an expanded third cell from the pore end. Three randomly selected isolates from different plates were applied in further analysis. The internal transcribed spacer (ITS: MZ026467) region, translation elongation factor (EF-1α: MZ042646), large subunit ribosomal RNA (LSU: MZ026469), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: OK086974) segments were amplified and sequenced using primers ITS1/ITS4, EF-1/EF-2, LR5F/ LROR, and gpd1/gpd2, respectively (White et al. 1990; O'Donnell 1998; Marin-Felix 2020). The four segments were identical among the three isolates, which shared the highest nucleotide identities (100% on ITS, LSU, and GAPDH, and 99.8% on EF-1α) with C. lunata strains in the GenBank database. Based on the ITS and GAPDH sequences, phylogenetic analysis using Maximum-likelihood and Bayesian inference methods by W-IQ-TREE (accessed on 09/01/2021) (Trifinopoulos et al., 2016) and MrBayes v3.2.7a (Ronquist et al., 2012) both supported that the isolates belong to C. lunata. For pathogenicity tests, we sprayed the conidial suspension (1×106 conidia/ml) of each of the three isolates on three healthy three-month-old Shatangju seedlings, which were kept under 27 °C and ~100% humidity in plastic bags in the greenhouse. Another three seedlings were sprayed with sterile water and kept under the same conditions as negative controls. Three days after inoculation, small maroon dots started appearing on the inoculated leaves, and the symptoms, same as those observed in the field, developed afterward. No symptoms appeared on the negative controls. C. lunata was reisolated from the infected leaves of all plants inoculated with the three isolates but not from the negative controls. This pathogen has been reported to cause diseases in Pennisetum hydridum and Capsicum frutescens in China (Xu et al. 2018; Pei et al. 2017). But to our knowledge, this is the first report of C. lunata causing leaf spots on citrus plants in the world.

Integrative analysis of metabolome and transcriptome profiles provides insight into the fruit pericarp pigmentation disorder caused by 'Candidatus Liberibacter asiaticus' infection.

BACKGROUND: Mandarin 'Shatangju' is susceptible to Huanglongbing (HLB) and the HLB-infected fruits are small, off-flavor, and stay-green at the maturity period. To understand the relationship between pericarp color and HLB pathogen and the effect mechanism of HLB on fruit pericarp coloration, quantitative analyses of HLB bacterial pathogens and carotenoids and also the integrative analysis of metabolome and transcriptome profiles were performed in the mandarin 'Shatangju' variety with four different color fruits, whole green fruits (WGF), top-yellow and base-green fruits (TYBGF), whole light-yellow fruits (WLYF), and whole dark-yellow fruits (WDYF) that were infected with HLB. RESULTS: the HLB bacterial population followed the order WGF > TYBGF > WLYF > WDYF. And there were significant differences between each group of samples. Regarding the accumulation of chlorophyll and carotenoid, the chlorophyll-a content in WGF was the highest and in WDYF was the lowest. The content of chlorophyll-b in WGF was significantly higher than that in other three pericarps. There were significant differences in the total content of carotenoid between each group. WGF and TYBGF pericarps were low in phytoene, γ-carotene, ß-cryptoxanthin and apocarotenal, while other kinds of carotenoids were significantly higher than those in WDYF. And WLYF was only short of apocarotenal. We comprehensively compared the transcriptome and metabolite profiles of abnormal (WGF, TYBGF and WLYF) and normal (WDYF, control) pericarps. In total, 2,880, 2,782 and 1,053 differentially expressed genes (DEGs), including 121, 117 and 43 transcription factors were identified in the three comparisons, respectively. The qRT-PCR confirmed the expression levels of genes selected from transcriptome. Additionally, a total of 77 flavonoids and other phenylpropanoid-derived metabolites were identified in the three comparisons. Most (76.65 %) showed markedly lower abundances in the three comparisons. The phenylpropanoid biosynthesis pathway was the major enrichment pathway in the integrative analysis of metabolome and transcriptome profiles. CONCLUSIONS: Synthesizing the above analytical results, this study indicated that different color pericarps were associated with the reduced levels of some carotenoids and phenylpropanoids derivatives products and the down-regulation of proteins in flavonoids, phenylpropanoids derivatives biosynthesis pathway and the photosynthesis-antenna proteins.

Antimicrobial Efficacy of Liposome-Encapsulated Citral and Its Effect on the Shelf Life of Shatangju Mandarin.

ABSTRACT: Liposome-encapsulated citral was prepared by means of a hot homogenization method. The microstructure, particle size, and zeta potential of the capsules were analyzed by transmission electron microscope and dynamic light scattering, respectively, in which the results showed a good dispersion stability of the citral-loaded liposome. In vitro tests showed that liposome-encapsulated citral significantly (P < 0.05) reduced the populations of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Penicillium italicum more than free citral. In vivo tests conducted on fresh Shatangju mandarin showed that liposome-encapsulated citral-treated fruit exhibited a decay incidence of 56.67%, which is 42.04% lower than free citral-treated fruit (97.78%) after 26 days of storage at 25°C and 60 to 70% relative humidity. Additionally, fruit treated with citral-loaded liposome significantly reduced weight loss and viable yeast and mold during storage. In summary, liposome-encapsulated citral could be an effective antimicrobial agent to extend the shelf life of the Shatangju mandarin.

Metabolomics Analysis of the Peels of Different Colored Citrus Fruits (Citrus reticulata cv. 'Shatangju') During the Maturation Period Based on UHPLC-QQQ-MS.

Citrus is a globally consumed fruit with great popularity. Mandarin (Citrus reticulata cv. 'Shatangju') is a local variety, and its planting area and yield are the greatest regarding fruit tree planting in Guangdong Province, China. However, its resistance to Huanglongbing (HLB) is weak. After infection by HLB, the fruits cannot develop normally. In this study, four kinds of fruits were classified as HBG, XQG, ZQG, and DHG, according to the color of their peels. The metabolomes of the three abnormally colored groups (HBG, XQG, and ZQG) and the normally colored group (DHG) were compared using a UPLC-QQQ-MS-based metabolomics approach. In total, 913 metabolites were identified and classified into 23 different categories, including phenylpropanoids and flavonoids; among them, 215 (HBG, 177; XQG, 124; and ZQG, 62) metabolites showed differential accumulation in the three comparison groups (HBG/XQG/ZQG versus DHG). A total of 2 unique metabolites, O-caffeoyl maltotriose and myricetin were detected only in DHG samples. When comparing HBG with DHG, there were 109 decreased and 68 increased metabolites; comparing XQG with DHG, there were 88 decreased and 36 increased metabolites; comparing ZQG with DHG, 41 metabolites were decreased, and 21 metabolites were increased. Metabolic pathway enrichment analysis of these differential metabolites showed significant enrichment of the "phenylpropanoid biosynthesis" pathway in all comparison groups. The hierarchical cluster analysis of the differential metabolites of the four groups showed a clear grouping patterns. The relative contents of three phenylpropanoids, four flavonoids, two alkaloids, one anthocyanin, and two other metabolites were significantly different between each comparison group. This study might provide fundamental insight for the isolation and identification of functional compounds from the peels of citrus fruit infected with HLB and for in-depth research on the effect of HLB on the formation of fruits pigment and the development of HLB-resistant citrus varieties.