Instituto Ludwig de Pesquisa sobre o Câncer, Rua Prof. Antonio Prudente, 109-4 andar, 01509-010, Sao Paulo-SP

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The genome sequence of the plant pathogen Xylella fastidiosa

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants.

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Xylella fastidiosa: Cause of Pierce's Disease of Grapevine and Other Emergent Diseases.

Numerous bacteria have been isolated from within plants, and many reported from xylem, but only three species of xylem-limited bacteria (XLB) that are fastidious in cultural requirements, are plant pathogens, and exclusively occupy xylem, have been well characterized. Two XLB, Xylella fastidiosa and Pseudomonas syzygii, are transmitted by sucking insects that feed on xylem sap but are not transmitted mechanically from plant to plant. In contrast, Clavibacter xyli is mechanically transmitted to plants by cutting tools. All of these XLB occupy a highly specialized yet diverse ecological niche: the water-conducting systems of an extremely wide range of plant hosts. A variety of detection methods are available as diagnostic aids; each method has advantages and disadvantages; no single method is best for all uses. Molecular and genetic comparisons of strains of XLB lag behind progress being made for many other plant-pathogenic bacteria, but such studies are needed to answer important questions: (a) How do XLB move from cell to cell within plants? (b) What are the physiological and genetic bases of plant host specificity for XLB? (c) Why are only xylem-feeding specialists vectors of X. fastidiosa (and probably P. syzygii), when many leafhoppers feed regularly (but not continuously) on xylem?

Fastidious xylem-limited bacterial plant pathogens

Xylella fastidiosa is a gram-negative bacterium limited to the plant xylem vessels, a unique, nutritionally dilute habitat (27). The bacterium is transmitted to new host plants during xylem sap feeding by insect vectors such as sharpshooter leafhoppers (Hemiptera, Cicadellidae) or spittlebugs (Hemiptera, Cercopidae) (30). X. fastidiosa multiplies and spreads from the site of infection to colonize the xylem, a water transport network of vessels composed of dead, lignified cells. Vessels are interconnected by channels, called bordered pits, that allow the passage of xylem sap but block the passage of larger objects due to the presence of a pit membrane (34). Bacterial cells attach to the vessel wall and multiply, forming biofilm-like colonies that can, when sufficiently large, completely occlude xylem vessels, blocking water transport (35). Many agriculturally important plants, such as citrus, almond, coffee, and grapevines, are susceptible to diseases caused by X. fastidiosa (20). In susceptible plants, leaf scorching, fruit shriveling, and other symptoms result, probably due to the increased stress of xylem blockage as colonization ensues. However, within the majority of host plants, X. fastidiosa behaves as a harmless endophyte (11, 31). X. fastidiosa population comparisons between grapevines resistant and susceptible to Pierce's disease (PD) demonstrate a positive correlation between high populations and symptom expression (12). However, we still lack an understanding of the process of colonization and what specific aspects of large populations of X. fastidiosa lead to symptom expression.

Many studies of X. fastidiosa colonization have involved grinding infected plant tissue and detecting bacteria by culture (1, 17), enzyme-linked immunosorbent assay (6, 21), or PCR (23); with these methods, the distribution of X. fastidiosa within the tissue was unknown. For example, it is not known whether large populations seen in plant macerates of susceptible plants correspond to many small communities or a few large communities of X. fastidiosa. However, the size, distribution, and behavior of X. fastidiosa colonies within the xylem network may be important factors in the generation of PD symptoms. Understanding the pattern of X. fastidiosa colonization of the xylem and relating this pattern to symptom expression will further our progress in understanding PD as well as the endophytic life-style of the pathogen. To this end, several other studies have analyzed X. fastidiosa in planta using various types of microscopy, including light microscopy (13, 19), scanning electron microscopy (35), transmission electron microscopy (24), and immunofluorescence microscopy (4).

In these studies, it was determined that the frequency of vessels colonized and blocked by X. fastidiosa in grapes is positively correlated with disease symptom development over the growing season (19) and within individual plants (35). X. fastidiosa colonies in the xylem were reported to be of different sizes(35), to be either distributed evenly throughout the vessel or appressed against the vessel wall (24), and sometimes to be accompanied by a matrix presumed to be a gel of either plant or bacterial origin; however, no quantitative analysis of these observations has been reported (4, 13, 24, 35). One drawback to the methods used in these studies is that they required extensive preparation of the sample prior to microscopy, such that only small regions of the plant could be examined. In addition, dissection and preparation of samples is fraught with procedural issues, leading to uncertainty about the original spatial distribution of the pathogen.

In this study, we characterized X. fastidiosa colonization of grapevine in a quantitative manner and related patterns of colonization to symptom development in the plant tissue. To conduct this analysis, we engineered a strain of X. fastidiosa that constitutively expresses a green fluorescent protein (Gfp). Fluorescent X. fastidiosa cells were visualized directly in the plant by confocal laser-scanning microscopy (CLSM). This type of microscopy captures images from within a sample, allowing visualization of unperturbed X. fastidiosa cells in intact xylem vessels. Fixation, washing, and staining are not needed, and sample dissection is minimal, eliminating the potential for artifacts that can affect other types of microscopy used for in planta analysis of X. fastidiosa. We chose to limit our study to petioles, where high levels of colonization of the xylem occur in symptomatic plants (19).

https://aem.asm.org/content/aem/69/12/7319.full.pdf

Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera.

A xylem-limited bacterium resemblingXylella fastidiosa has been shown previously by electron mmcroscopy to be associated with citrus variegated chlorosis (CVC), a new disease of sweet organe tress in Brazil. A bacterium was consistently cultured from plant tissues from CVC twigs of sweet orange trees but not from tissues of healthy trees on several cell-free media known to support the growth ofXylella fastidiosa. Bacterial colonies typical ofX. fastidiosa became visible on PW, CS20, and PD2 agar media after 5 and 7–10 days of incubation, respectively. The cells of the CVC bacterium were rod-shaped, 1.4–3 μm in length, and 0.2–0.4 μm in diameter, with rippled walls. An antiserum against an isolate (8.1.b) of the bacterium gave strong positive reactions to double-antibody-sandwich (DAS), enzyme-linked immunosorbent assay (ELISA) with other cultured isolates from CVC citrus, as well as with several type strains ofX. fastidiosa. This result indicates that the CVC bacterium is a strain ofX. fastidiosa. ELISA was also highly positive with all leaves tested from CVC-affected shoots. Leaves from symptomless tress reacted negatively. Sweet organe seedlings inoculated with a pure culture of the CVC bacterium supported multiplication of the bacterium, which became systemic with 6 months after inoculation and could be reisolated from the inoculated seedlings. Symptoms characteristic of CVC developed 9 months post inoculation.

Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain ofXylella fastidiosa.

Victoria Rossetti

Papers

Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain ofXylella fastidiosa.