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.

Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.

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.

Citrus Huanglongbing (HLB) is a serious disease for the citrus industry. Earlier studies showed that ampicillin (Amp) can reduce titers of the pathogen which causes HLB and the bacteria Candidatus Liberibacter asiaticus (CLas) in HLB-affected citrus. CLas has not yet been cultured, so the mechanisms of Amp against CLas are unclear. Some chemicals were demonstrated to trigger citrus defense systems against CLas. Therefore, we hypothesize that Amp may induce citrus defenses against CLas. Here we applied three nano-formulations of varying droplet sizes to HLB-affected citrus to achieve different accumulated concentrations of Amp (high, medium, low) in the plants. We then used RNA-seq to analyze induction of gene expression of citrus defense systems against CLas in response to different concentrations of Amp. The results indicated that at all accumulated concentrations of Amp can significantly suppress CLas titer and mitigate HLB symptoms. Transcriptomic analyses showed that Amp treatment induced expression of heat shock proteins (Hsps) in HLB-affected citrus, and these Hsps were significantly related to several defense genes encoding R proteins, transcription factors, splicing factors, RNA-binding proteins, RNA-dependent RNA polymerase, Gibberellic acid methyltransferase 2, L-ascorbate peroxidase 2, and ferruginol synthase that confer tolerance to CLas in citrus plants. Taken together, these results suggest that Amp treatment of citrus plants can trigger expression of Hsps and related defense genes to respond to CLas infection. These findings are valuable for developing novel strategies to combat citrus HLB.

Citron is a medicinal and edible plant. Terpenoids exhibit a wide range of biological activities. However, terpenoids in citron remain underexplored. In this study, a widely targeted metabolomics approach was employed to analyze the accumulation and variation patterns of terpenoid metabolites in citron flavedo at six developmental stages (50, 80, 110, 140, 170, and 210 d after flowering). The results showed that a total of 215 metabolites were detected, with monoterpenes (90) and triterpenes (51) being the major ones, accounting for 41.86% and 23.72% of the total, respectively. A total of 67 differential terpenoid metabolites were identified across different developmental stages. The number of up-regulated metabolites consistently exceeded that of down-regulated metabolites in each comparison group. Bitterness-related triterpenoid metabolites such as limonin, nomilin, obacunone, nomilinic acid, and obacunoic acid peaked at 170 d after flowering but exhibited an overall downward trend at 210 d. Most of the limonoid metabolites were significantly or extremely significantly positively correlated with one another; notably, nomilin displayed strong positive correlations with obacunone, deacetylnomilin, nomilinic acid, and obacunoic acid. This study delivers the first in-depth analysis of terpenoids in citron throughout development stages, laying a foundation for ongoing research and development of terpenoids in citron.