Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of “Candidatus Liberibacter asiaticus” in planta

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide.

黄龙病（Huanglongbing，简称HLB，又称柑橘青果病）被认为是全球范围内对柑橘产业造成严重破坏的最具毁灭性病害。HLB由亚洲韧皮部杆菌（Candidatus Liberibacter asiaticus，缩写CLas）引起，这是一种难以培养的韧皮部限制性革兰氏阴性细菌，传播媒介为亚洲柑橘木虱。截至目前，尚无针对CLas的有效防治手段。为减轻HLB对产业的影响并保障柑橘种植户的利益，亟需筛选或开发能够抑制或清除感病柑橘植株内CLas的抑菌分子。本研究首次证实了两种抗菌化合物及其组合对CLas的植物体内防控效果，这两种化合物分别为2S白蛋白（2S albumin，一种植物源蛋白，分子量约12.5 kDa）、纳米氧化锌（Nano-Zinc Oxide，缩写Nano-ZnO，直径约4.0 nm）。将这些化合物的水基制剂通过树干注射法施用于感染HLB的莫姆比甜橙（Citrus sinensis）植株，该植株以3年生粗柠檬（C. jambhiri）作为砧木，且在无虫隔离网室中维持已知的CLas滴度水平。本研究采用体积比为1:1的2S白蛋白（330 ppm）与纳米氧化锌（330 ppm）混合制剂。处理后每30天（DAT，即处理后天数）通过TaqMan定量聚合酶链式反应（TaqMan-qPCR）评估施药植株的CLas病原载荷动态变化，监测周期直至处理后120天。结果显示，2S白蛋白-纳米氧化锌混合制剂在所有处理组中效果最优，在处理后30、60、90及120天，其CLas种群数量较初始菌量（每12.5 ng基因组DNA）分别下降96.2%、97.6%、95.6%及97%。本研究证实了2S白蛋白-纳米氧化锌混合制剂作为抗菌药剂在植物体内抑制CLas的有效性，其有望开发为新型抗CLas治疗手段，以减轻全球范围内HLB对柑橘产业造成的危害。