John S. Hartung

Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis

By cloning and sequencing specific randomly amplified polymorphic DNA (RAPD) products, we have developed pairs of PCR primers that can be used to detect Xylella fastidiosa in general, and X. fastidiosa that cause citrus variegated chlorosis (CVC) specifically. We also identified a CVC-specific region of the X. fastidiosa genome that contains a 28-nucleotide insertion, and single base changes that distinguish CVC and grape X. fastidiosa strains. When using RAPD products to develop specific PCR primers, we found it most efficient to screen for size differences among RAPD products rather than presence/absence of a specific RAPD band.

Coffee leaf scorch bacterium: axenic culture, pathogenicity, and comparison with Xylella fastidiosa of citrus.

Symptoms of coffee leaf scorch (CLS) appear on young flushes of field plants as large marginal and apical scorched areas on recently mature leaves. Affected leaves drop, shoot growth is stunted, and apical leaves are small and chlorotic. Symptoms may progress to shoot dieback. Only scorched leaves which could not be related to other known agents consistently contained bacteria and bacterial agglomerates when observed with light microscopy. Only plants with these symptoms were positive in enzyme-linked immunosorbent assay (ELISA) tests using antiserum to Xylella fastidiosa Wells et al. The bacterium Xylella fastidiosa Wells et al. was isolated in November 1995 from coffee (Coffea arabica) leaves with scorch symptoms on supplemented periwinkle wilt medium. Colonies were circular, dome-shaped, white, and 0.5 to 1.5 mm in diameter. Two of 10 young coffee seedlings stem-inoculated with a suspension of the isolated X. fastidiosa in January 1996 showed leaf scorch symptoms 3 to 5 months later, contained bacteria in xylem extracts, and reacted positively in ELISA using antiserum to the citrus variegated chlorosis (CVC) strain of X. fastidiosa. ELISA-positive bacteria were reisolated from this plant. None of the symptomless plants, including controls, revealed bacteria on microscopic examinations, ELISA, or isolation attempts. Antisera developed against cultured bacteria from both CLS and CVC plants reacted positively against plant extracts of both diseases in dot immunobinding assays (DIBA). The level of detection was about 5 × 105 bacteria ml-1 for both homologous and heterologous reactions. The polymerase chain reaction amplification products produced by CLS and CVC strains of X. fastidiosa were indistinguishable. Geographical distribution of these strains is not the same. CLS is widespread and usually occurs if coffee is adjacent to CVC-affected citrus. However, CVC does not always occur when citrus is grown adjacent to CLS-affected coffee. The bacteria are closely related, if not identical.

Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data

Genetic relationships among 11 Xylella fastidiosa strains isolated from mulberry, almond, ragweed, grape, plum, elm, and citrus were determined by random amplified polymorphic DNA (RAPD). Twenty-two 10-base primers amplified a total of 77 discrete polymorphic bands. Phenetic analysis based on a similarity matrix corresponded well with previous reports on X. fastidiosa RFLP-based similarity relationships, indicating that RAPD-PCR amplification products can be used as a reliable indicator of genetic distance in X. fastidiosa. Cladistic analysis suggests the existence of five groups of X. fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and the mulberry group.

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two‐step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X. fastidiosa. A total of 347 leafhoppers representing 16 species were captured and sampled from American elm (Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta, regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa. Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.

Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR

Abstract. Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierces Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0.872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Distribution of Xylella fastidiosa in citrus rootstocks and transmission of citrus variegated chlorosis between sweet orange plants through natural root grafts

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Citrus and coffee strains of Xylella fastidiosa induce Pierce's disease in grapevine.

Xylella fastidiosa causes citrus variegated chlorosis (CVC) disease in Brazil and Pierces disease of grapevines in the United States. Both of these diseases cause significant production problems in the respective industries. The recent establishment of the glassy-winged sharpshooter in California has radically increased the threat posed by Pierces disease to California viticulture. Populations of this insect reach very high levels in citrus groves in California and move from the orchards into the vineyards, where they acquire inoculum and spread Pierces disease in the vineyards. Here we show that strains of X. fastidiosa isolated from diseased citrus and coffee in Brazil can incite symptoms of Pierces disease after mechanical inoculation into seven commercial Vitis vinifera varieties grown in Brazil and California. Thus, any future introduction of the CVC strains of X. fastidiosa into the United States would pose a threat to both the sweet orange and grapevine industries. Previous work has clearly shown that the strains of X. fastidiosa isolated from Pierces disease- and CVC-affected plants are the most distantly related of all strains in the diverse taxon X. fastidiosa. The ability of citrus strains of X. fastidiosa to incite disease in grapevine is therefore surprising and creates an experimental system with which to dissect mechanisms used by X. fastidiosa in plant colonization and disease development using the full genome sequence data that has recently become available for both the citrus and grapevine strains of this pathogen.

Genetic diversity of citrus bacterial canker pathogens preserved in herbarium specimens

Citrus bacterial canker (CBC) caused by Xanthomonas axonopodis pv. citri (Xac) was first documented in India and Java in the mid 19th century. Since that time, the known distribution of the disease has steadily increased. Concurrent with the dispersion of the pathogen, the diversity of described strains continues to increase, with novel strains appearing in Saudi Arabia, Iran, and Florida in the last decade. Herbarium specimens of infected plants provide an historical record documenting both the geographic distribution and genetic diversity of the pathogen in the past. However, no method was available to assess the genetic diversity within these herbarium samples. We have developed a method, insertion event scanning (IES), and applied the method to characterize the diversity present within CBC populations documented as herbarium specimens over the past century. IES is based on the specific amplification of junction fragments that define insertion events. The potential for IES in current forensic applications is demonstrated by finding an exact match of pathogen genotypes preserved in herbarium specimens from Japan and Florida, demonstrating the source of the original outbreak of citrus canker in Florida in 1911. IES is a very sensitive technique for differentiating bacterial strains and can be applied to any of the several hundred bacteria for which full genomic sequence data are available.