Multiplication and Movement of a Citrus Strain ofXylella fastidiosaWithin Sweet Orange

Abstract

Citrus variegated chlorosis (CVC), caused by the xylem-limited bacterium Xylella fastidiosa, has arisen from obscurity to become a major threat to the Brazilian citrus industry in less than 10 years. Since X. fastidiosa was first shown to cause CVC (3,20), serological (5) and molecular methods (1,21,25) have been developed to detect the bacterium in citrus. The pathogen is naturally transmitted to citrus by sharpshooter (Cicadellinae) leafhoppers (29). Transmission by natural root grafts and by top grafting with infected budsticks also were reported (10). Epidemiological studies suggest that most spread of CVC is from tree to tree within the citrus groves (19). Therefore, management of CVC is mainly based on planting healthy nursery trees, vector control, and removal of inoculum by roguing of symptomatic young plants (<2 years old) and pruning of branches with early foliar symptoms in older trees (8,30). The population behavior of X. fastidiosa within citrus is crucial to its pathology and control, but bacterial colonization of citrus has not been studied quantitatively and some characteristics of infection, movement of X. fastidiosa within citrus, and disease development remain unknown. He et al. (10) used polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) to detect movement of X. fastidiosa from scion graft to rootstock but did not quantify populations of the pathogen.A limiting factor to studies concerning X. fastidiosa is the slow growth of the pathogen on solid medium, leading to frequent contamination of culture attempts. This has hindered attempts to quantify populations of X. fastidiosa. Hopkins (14) developed a protocol to estimate population of viable X. fastidiosa cells from grapevines with Pierce's disease (PD) based on counting CFU of this bacterium on solid media by dilution plating. A modification of this technique (11) was used to study PD strains of X. fastidiosa in alternative hosts, with the bacterium reaching high populations (log 8 CFU/g of plant tissue) in grapevine, 100-fold lower populations in an asymptomatic host, Himalayan blackberry, and still lower, nonsystemic populations in water grass and mugwort. Populations of X. fastidiosa estimated by dilution plating were also correlated with vector transmission rates; bacterial populations lower than log 6 to log 7 CFU/g determined by culture in grapevine resulted in reduced transmission efficiency (13).The objective of our study was to follow the fate of a CVC strain of X. fastidiosa in citrus seedlings under greenhouse conditions and to estimate population levels of X. fastidiosa in symptomatic leaves of infected citrus trees in commercial orchards. We used culturing to quantify X. fastidiosa in leaves collected from the field and its growth and movement within citrus seedlings, following mechanical inoculation in the stem and leaf petioles with variable concentrations of live cells. We also tested different media for X. fastidiosa growth and evaluated citrus tissue homogenates for inhibition of bacterial growth i...

Intercepted isolates of Xylella fastidiosa in Europe reveal novel genetic diversity

“…Xylella fastidiosa was cultured from all host plants during the Spring and Summer of 2015, except for samples CO15 and COBZ, using standard protocols (Almeida et al 2001;Cariddi et al 2014) and triply cloned prior to recovering the DNA for the amplification and sequencing of the set of genes part of the MLST scheme (leuA, petC, malF, cysG, holC, nuoL, and gltT; Yuan et al 2010). MLST data for CO15 and COBZ were obtained via direct PCR of total DNA extracted from infected plant material; DNA was extracted using CTAB buffer following the procedure reported by Loconsole et al (2014).…”

Spatial Genetic Structure of Coffee-Associated Xylella fastidiosa Populations Indicates that Cross Infection Does Not Occur with Sympatric Citrus Orchards

“…All X. fastidiosa populations were established throughout the year 2012 by sampling X. fastidiosa-symptomatic tissues from coffee and citrus trees according to symptoms as described by de Lima et al (1998) andde Souza et al (2009), respectively. Bacteria were isolated from both symptomatic branches (3 mm in diameter) or leaf petioles on solid buffered charcoal yeast extract (BCYE) agar medium (Wells et al 1981), as previously described (Almeida et al 2001;Coletta-Filho and Machado 2003). Solid media plates were incubated at 28°C for at least 15 days before appearance of colonies, which were identified as X. fastidiosa based on fastidious growth, coloring, and polymerase chain reaction (PCR) assays using primers specific to X. fastidiosa subsp.…”

“…All the strains used for inoculation in coffee and citrus were also inoculated in Nicotiana tabacum as a control for cell viability during inoculation and persistence of infection over time, because tobacco is highly susceptible to X. fastidiosa (Lopes et al 2000). Seedlings of all tested hosts-C. sinensis Pera, Coffea arabica (L.) Mundo Novo, and Nicotiana tabacum 'Havana'-were inoculated in two different points of the main stem with 10 µl of X. fastidiosa suspensions at 10 8 CFU/ml, following methods previously described (Almeida et al 2001). The plants were maintained inside an insect-free greenhouse.…”

Transformação genética de laranja doce (Citrus sinensis L. Osbeck) com o gene cecropin MB39 e avaliação de plantas transgênicas inoculadas com Xylella fastidiosa Wells et al.

“…A velocidade da movimentação parece ser influenciada pela estirpe da bactéria, já que Fry e Milholland (1990) verificaram que estirpe virulenta, em pecíolos e ramos de videira, colonizou o 17º internódio acima do local de inoculação em apenas oito semanas, já a estirpe avirulenta permaneceu no 10º internódio. Quanto à distribuição da bactéria pela planta, Almeida et al (2001) verificaram que a bactéria apresentou translocação ascendente e descendente, sendo encontrada nas raízes das plantas de citros quando inoculadas na parte aérea.…”

“…No isolamento primário e quantificação da população bacteriana das plantas de citros inoculadas, utilizou-se o método de isolamento em meio de cultura descrito por Hill e Purcell (1995b) adaptado por Almeida et al (2001).…”