Sauer Aline Vanessa, Zanutto Carlos Alexandre, Nocchi Paula Thais Requena, Machado Marcos Antonio, Bock Clive H, Nunes William M C
Núcleo de Pesquisa em Biotecnologia Aplicada (NBA), Universidade Estadual de Maringá (UEM) 87020-900, Maringá, Paraná, Brazil.
Centro de Citricultura "Sylvio Moreira", Instituto Agronomico de Campinas, 13490-970, Cordeirópolis, SP, Brazil.
Plant Dis. 2015 Aug;99(8):1125-1132. doi: 10.1094/PDIS-09-14-0926-RE. Epub 2015 Jun 12.
Huanglongbing (HLB) is considered one of the most destructive diseases of citrus because the plants rapidly become unproductive, enter a decline, and eventually die. HLB is caused by the phloem-limited bacterium 'Candidatus Liberibacter' spp. The objective of this study was to evaluate seasonal variation of the in planta population of 'Ca. Liberibacter asiaticus' in the foliage of citrus trees in Brazil using real-time polymerase chain reaction (qPCR). Eleven plants (naturally infected, then screened) in the field with very mild and localized symptoms of HLB were confirmed to be 'Ca. L. asiaticus' infected by conventional PCR, and the canopies were divided into four quadrants. The bacterial population in the trees was tested on a monthly basis for up to 20 months by quantifying 'Ca. L. asiaticus' DNA using qPCR 'Ca. L. asiaticus'-specific primers (As84F/As180R). The average cycle threshold (C) values, which relate to 'Ca. L. asiaticus' titer, were analyzed using a mixed model. Significant differences were observed in C values between seasons (F = 8.77, P = 0.0004), and abrupt changes were observed in C values in different quadrants of the trees. Autumn had the lowest C values, indicating the highest 'Ca. L. asiaticus' titer, and, thus, is considered the best period to detect 'Ca. L. asiaticus' infection in foliage of citrus trees in southern Brazil. In addition to the seasonal changes in C values, there was an initial decline in the C value in the months following initial detection, the rate of decline slowing with time. Concomitant with the increase of the bacterial population in the host, there was an increase in severity of HLB symptoms in the trees over time (Spearman's rank correlation, r = -0.4083, P < 0.0001). The results identify the optimal season to sample foliage for 'Ca. L. asiaticus' in southern Brazil (autumn) and confirm the importance of sample timing to maximize detection of 'Ca. L. asiaticus' and, thus, contribute to the search for effective measures to manage HLB.
柑橘黄龙病（HLB）被认为是柑橘最具破坏性的病害之一，因为染病植株会迅速减产，进入衰退期，最终死亡。HLB由韧皮部限制性细菌“Ca. Liberibacter”属引起。本研究的目的是使用实时聚合酶链反应（qPCR）评估巴西柑橘树叶片中“Ca. Liberibacter asiaticus”在植株内的种群季节性变化。对田间11株有非常轻微和局部HLB症状（自然感染，随后筛选）的植株进行常规PCR检测，确认感染了“Ca. L. asiaticus”，并将树冠分为四个象限。通过使用qPCR“Ca. L. asiaticus”特异性引物（As84F/As180R）定量“Ca. L. asiaticus”DNA，每月对树中的细菌种群进行检测，持续20个月。使用混合模型分析与“Ca. L. asiaticus”滴度相关的平均循环阈值（C）值。在不同季节的C值之间观察到显著差异（F = 8.77，P = 0.0004），并且在树的不同象限中观察到C值的突然变化。秋季的C值最低，表明“Ca. L. asiaticus”滴度最高，因此被认为是在巴西南部柑橘树叶片中检测“Ca. L. asiaticus”感染的最佳时期。除了C值的季节性变化外，在初次检测后的几个月中C值最初下降，下降速率随时间减缓。随着宿主中细菌种群的增加，树中HLB症状的严重程度随时间增加（斯皮尔曼等级相关性，r = -0.4083，P < 0.0001）。结果确定了在巴西南部（秋季）对“Ca. L. asiaticus”进行叶片采样的最佳季节，并证实了采样时间对于最大限度地检测“Ca. L. asiaticus”的重要性，从而有助于寻找管理HLB的有效措施。
