Magnesium (Mg)-deficiency occurs most frequently in strongly acidic, sandy soils. Citrus are grown mainly on acidic and strong acidic soils. Mg-deficiency causes poor fruit quality and low fruit yield in some Citrus orchards. For the first time, we investigated Mg-deficiency-responsive miRNAs in ‘Xuegan’ (Citrus sinensis) roots using Illumina sequencing in order to obtain some miRNAs presumably responsible for Citrus Mg-deficiency tolerance. We obtained 101 (69) miRNAs with increased (decreased) expression from Mg-starved roots. Our results suggested that the adaptation of Citrus roots to Mg-deficiency was related to the several aspects: (a) inhibiting root respiration and related gene expression via inducing miR158 and miR2919; (b) enhancing antioxidant system by down-regulating related miRNAs (miR780, miR6190, miR1044, miR5261 and miR1151) and the adaptation to low-phosphorus (miR6190); (c) activating transport-related genes by altering the expression of miR6190, miR6485, miR1044, miR5029 and miR3437; (d) elevating protein ubiquitination due to decreased expression levels of miR1044, miR5261, miR1151 and miR5029; (e) maintaining root growth by regulating miR5261, miR6485 and miR158 expression; and (f) triggering DNA repair (transcription regulation) by regulating miR5176 and miR6485 (miR6028, miR6190, miR6485, miR5621, miR160 and miR7708) expression. Mg-deficiency-responsive miRNAs involved in root signal transduction also had functions in Citrus Mg-deficiency tolerance. We obtained several novel Mg-deficiency-responsive miRNAs (i.e., miR5261, miR158, miR6190, miR6485, miR1151 and miR1044) possibly contributing to Mg-deficiency tolerance. These results revealed some novel clues on the miRNA-mediated adaptation to nutrient deficiencies in higher plants.

Boron (B) toxicity in Citrus is a common physiological disorder leading to reductions in both productivity and quality. Studies on how Citrus roots evade B toxicity may provide new insight into plant tolerance to B toxicity. Here, using Illumina sequencing, differentially expressed microRNAs (miRNAs) were identified in B toxicity-treated Citrus sinensis (tolerant) and C. grandis (intolerant) roots. The results showed that 37 miRNAs in C. grandis and 11 miRNAs in C. sinensis were differentially expressed when exposed to B toxicity. Among them, miR319, miR171, and miR396g-5p were confirmed via 5′-RACE and qRT-PCR to target a myeloblastosis (MYB) transcription factor gene, a SCARECROW-like protein gene, and a cation transporting ATPase gene, respectively. Maintenance of SCARECROW expression in B treated Citrus roots might fulfill stem cell maintenance, quiescent center, and endodermis specification, thus allowing regular root elongation under B-toxic stress. Down-regulation of MYB due to up-regulation of miR319 in B toxicity-treated C. grandis roots might decrease the number of root tips, thereby dramatically changing root system architecture. Our findings suggested that miR319 and miR171 play a pivotal role in Citrus adaptation to long-term B toxicity by targeting MYB and SCARECROW, respectively, both of which are responsible for root growth and development.

Summary: The mechanisms underlying plant tolerance to boron (B) excess are far from fully understood. Here we characterized the role of the miR397‐CsiLAC4/CsiLAC17 (from Citrus sinensis) module in regulation of B flow. Live‐cell imaging techniques were used in localization studies. A tobacco transient expression system tested modulations of CsiLAC4 and CsiLAC17 by miR397. Transgenic Arabidopsis were generated to analyze the biological functions of CsiLAC4 and CsiLAC17. CsiLAC4’s role in xylem lignification was determined by mRNA hybridization and cytochemistry. In situ B distribution was analyzed by laser ablation inductively coupled plasma mass spectrometry. CsiLAC4 and CsiLAC17 are predominantly localized in the apoplast of tobacco epidermal cells. Overexpression of CsiLAC4 in Arabidopsis improves the plants’ tolerance to boric acid excess by triggering high‐B‐dependent lignification of the vascular system’s cell wall and reducing free B content in roots and shoots. In Citrus, CsiLAC4 is expressed explicitly in the xylem parenchyma and is modulated by B‐responsive miR397. Upregulation of CsiLAC4 in Citrus results in lignification of the xylem cell walls, restricting B flow from xylem vessels to the phloem. CsiLAC4 contributes to plant tolerance to boric acid excess via high‐B‐dependent lignification of cell walls, which set up a ‘physical barrier’ preventing B flow.

Several tephritid fruit fly species that are damaging to citrus in China are world-wide quarantine pests. Two field tests were conducted in China to evaluate the effectiveness of fruit bagging (i.e., fruits were grown in bags for at least 1.5 mo until harvest) and culling for risk mitigation of these pests during the fruit harvest season of 2017. The first test was conducted in Pinghe County of Fujian Province. The purpose of this test was to assess the effectiveness of fruit bagging on risk mitigation of fruit flies affecting pomelo, Citrus maxima (Burm. fil.) Osbeck (Rutaceae). External inspection and internal fruit cutting of 3,000 bagged and 3,040 unbagged fruits revealed few oviposition marks and absence of living flies in the bagged fruits, compared to 129 fruit fly-infested fruits containing 634 live larvae and 4 pupae in the unbagged fruits. Later molecular and morphological identification concluded that these larvae and pupae were Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). The second test was conducted in Shimen County of Hunan Province. The purpose of this test was to assess the effectiveness of packing house culling on risk mitigation of fruit flies in Satsuma mandarin, Citrus unshiu (Swingle) Marcov. (Rutaceae). A total of 20,000 fruits were cut (10,000 fruits before culling, and another 10,000 after culling). In the fruits that did not receive the culling treatment, 1 infested fruit with 7 third instar larvae of Bactrocera minax (Enderlein) (Diptera: Tephritidae) and 25 fruits with fruit fly oviposition marks were found. In the fruits that received the culling treatment, fruit flies were absent and 10 fruits with oviposition marks were found. These results suggest that fruit bagging and packinghouse culling could contribute to risk mitigation of fruit flies in citrus in China. This is a preliminary report, with further work necessary to develop a systems approach for risk mitigation of fruit flies in the commodities.

Grapes are among the most widely cultivated fruit crops globally, yet their growth and yield are severely compromised by Colletotrichum viniferum, which causes a devastating disease that affects grape berries. The wall-associated kinase (WAK) gene family, a unique subfamily of receptor-like-kinases (RLKs), plays important roles in mediating plant responses to both abiotic and biotic stresses. However, the expression patterns and biological functions of grape WAKs in response to C. viniferum infection remain largely uncharacterized. In this study, a total of 57 VdWAK genes were identified and phylogenetically classified into twelve subgroups. Chromosomal localization and collinearity analyses further revealed that tandem duplication and segmental duplication events contributed to the expansion of the VdWAK gene family. Transcriptomic profiling identified VdWAK19 as a key responsive gene. It was predominantly expressed in mature berries but transcriptionally repressed upon C. viniferum infection. Virus-induced gene silencing assays in grape berries demonstrated that knockdown of VdWAK19 significantly reduced fruit resistance to C. viniferum infection. Overall, these findings advance our understanding of the functional roles of VdWAK genes during C. viniferum infection and provide a theoretical basis for the potential application of VdWAK19 in breeding grape varieties with enhanced resistance to ripe rot.