Plant Science

Citrus greening disease was first reported in Saudi Arabia during the 1970s when characteristic foliar and fruit symptoms were observed in commercial citrus groves. However, ‘Candidatus Liberibacter asiaticus’ (CLas) was not detected in symptomatic trees until 1981 to 1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the 13 citrus-growing regions of the country. Citrus trees were surveyed for the presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults revealed that trees were positive for CLas detection in 10 of the 13 survey regions; however, CLas was undetectable in ACP adults. Phylogenetic and single nucleotide polymorphism (SNP) analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (∼1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, U.S.A., isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.

Citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which was suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested to a useful strategy to reduce HLB damages. To provide the information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). ROS levels were consistently higher in symptomatic or asymptomatic branches than that in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared to healthy controls. This study sheds light on the pathogenicity of CLas by providing useful information on temporal dynamics of ROS production, cell death, and CLas growth within the phloem tissues.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, “Candidatus Liberibacter asiaticus” (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC–MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC.

Abstract Candidatus Liberibacter asiaticus (Las) is one of the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide. Due to the intracellular lifestyle and significant genome reduction, culturing Las in vitro has proven to be extremely challenging. In this study, we optimized growth conditions and developed a semi-selective medium based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the LG liquid medium with ca.100- to 1000-fold increase, which peaked after 4 to 6 weeks and were estimated to contain 106 to 107 cells/ml. The cultured Las bacteria remained in a dynamic state of growth for over 20 months and displayed limited growth in subcultures. The survival and growth of Las was confirmed by fluorescence in situ hybridization with Las-specific probes and expression of its metabolic genes. Growth of Las in the optimized medium relied on the presence of a helper bacterium, Stenotrophomonas maltophilia FLMAT-1 that is multi-drug resistant and dominant in the Las co-culture system. To recapitulate the disease, the co-cultured Las was inoculated back to citrus seedlings via psyllid feeding. Although the Las-positive rate of the fed psyllids and inoculated plants were relatively low, this is the first demonstration of partial fulfillment of Koch’s postulates with significant growth of Las in vitro and a successful inoculation of cultured Las back to psyllids and citrus plants that resulted in HLB symptoms. These results provide new insights into Las growth in vitro and a system for improvement towards axenic culture and anti-Las compound screening.