Isolation and Identification of Xanthomonas SP. from Infected Sample

Definition of Xanthomonas SP. and Xanthomonas citri

Xanthomonas citri is a bacterial pathogen that causes citrus canker (a disease which results in heavy economic losses to the citrus industry worldwide either in terms of damage to trees, particularly reduced fruit production), reduced access to export markets, or the costs of its prevention and control.

Lesions appear on leaves, twigs and fruit which cause defoliation, premature fruit abscission and blemished fruit, and can eventually kill the tree. It is introduced to new areas through the movement of infected citrus fruits and seedlings, and inadvertent re-introduction is highly likely despite the quarantine restrictions that are in place in many countries.

Locally, X. citri is rapidly disseminated by rainwater running over the surfaces of lesions and splashing onto uninfected shoots; spread is therefore greatest under conditions of hight temperature, heavy rainfall and strong winds. Some areas of the world have eradicated citrus canker, others have on-going eradication programmes, however, this pathogen remains a threat to all citrus-growing regions.

Taxonomic Tree of Xanthomonas Citri

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Xanthomonadales

Family: Xanthomonadaceae

Genus: Xanthomonas

Species: Xanthomonas citri

Xanthomonas citri is a Gram-negative, straight, rod-shaped bacterium measuring 1.5-2.0 x 0.5 0.75 μm. It is motile by means of a single and polar flagellum. It shares many physiological and biochemical properties with other members of the genus Xanthomonas.
It is chemo-organotrophic and obligately aerobic with the oxidative metabolism of glucose. Colonies are formed on nutrient agar (NA) plates containing glucose and are creamy-yellow with copious slime. The yellow pigment is xanthomonadin.
Catalase is positive, but Kovacs’ oxidase is negative or weak.

Nitrate reduction test is negative. Asparagine is not used as a sole source of carbon and nitrogen simultaneously; various carbohydrates and organic acids are as a sole source of carbon.
Hydrolysis of starch, casein, Tween 80 and aesculin is positive. Gelatine and pectate gel are liquefied. Growth requires methionine or cysteine and is inhibited by 0.02% triphenyltetrazolium chloride.
Biovars may be distinguished by utilization of mannitol. For further information on the bacteriological properties of X. citri, see Goto (1992).

Strains of groups B, C and D have many properties in common with group A, the differences being detected by the utilization of only a few carbohydrates (Goto et al.1980).

Symptoms of Canker

Canker lesions begin as light yellow, raised, spongy eruptions on the surface of leaves, twigs and fruits.
The lesions continuously enlarge from pin-point size over several months and can be of many different sizes based on the age of the lesion. As the lesions enlarge, the spongy eruptions begin to collapse, and brown depressions appear in their central portion, forming a crater-like appearance.

The edges of the lesions remain raised above the surface of host tissue and the area around the raised portion of the lesion may have a greasy appearance. The lesions become surrounded by characteristic yellow halos.
Canker lesions retain the erupted and spongy appearance under dry conditions, such as in a greenhouse; whereas they quickly enlarge and turn to flat lesions with a water-soaked appearance with frequent rain.
Canker lesions vary in maximum size from 5 to 10 mm, depending on the susceptibility of the host plant. The symptoms are similar on leaves, fruit and stems.

Canker lesions are histologically characterized by the development of a large number of hypertrophic cells and a small number of hyperplastic cells.
At an early stage of infection, the cells increase in size and the nuclei and nucleoids stain more easily; there is also an increase in the amount of cytoplasm synchronized with rapid enlargement.
However, these hypertrophied cells do not divide; cell division is only detected in the peripheral areas of lesions adjacent to healthy tissue.

Asiatic citrus canker is relatively easy to diagnose because of the characteristic symptoms of the disease. The pathogen may be isolated on nutrient agar (NA) or potate dextrose agar (PDA) at pH 6.8. Growth develops in 48 to 72 hours and colonies are creamy-yellow (on PDA) to straw-yellow (on NA).
The identification of isolated bacteria can be confirmed by pathogenicity to grapefruit leaves or by the polymerase chain reaction (PCR) using specific primers (Goto, 1992). Differentiation of canker A strains from other groups can be made with confidence using the methods reported in molecular characterization.
The diagnosis of citrus canker using polymerase chain reaction (PCR) is accurate and rapid (Wang et al. 2004: Mavrodieva et al., 2004). Golmohammadi et al. (2007) and Yin et al. (2007) found real-time PCR to be more effective at detecting X. citri, and up to 100-1000 times as sensitive.

Detection and Inspection of Xanthomonas Citri

Methods of detecting X. citri from natural habitats include leaf-infiltration, bacteriophage, fluorescent antibody and ELISA (Goto, 1992). The polymerase chain reaction and Dot blot immunobinding assay (DIA) were developed for rapid, sensitive, and specific detection of the pathogen. The detectable limits were reported to be around 30 c.f.u./ml for the former and 1000 c.f.u./ml for the latter (Hartung et al., 1993, 1996; Wang et al., 1997; Miyoshi et al., 1998).

Similarities to Other Species/Conditions

Citrus bacterial spot, caused by group E strains (X. campestris pv. citrumelo) (Schoulties et al., 1987) and bacterial brown spot, caused by Pseudomonas syringae (Shigeta and Nakata, 1995) are distinguished from citrus canker by a distinctive water-soaked appearance along the margins of lesions and the absence of hypertrophic eruption.
Canker B, C and D (Namekata, 1971) are distinguished by a narrow host range of mexican lime and lemon under natural conditions. Care must be taken not to confuse X. citri with Pantoea agglomerans in culture, P. agglomerans often produces yellow colonies on isolation media.

Prevention and Control of X. citri

Regulatory Control

Safeguards for exporting citrus fruits into the USA from Japan include: the establishment of isolated canker-free export areas; inspection of fruit by plant pathologists in both countries during harvesting and packing operations; pre-shipping surface sterilization with bactericidal dip; pre-shipping inspection using the bacteriophage method to ensure fruits are free of X. citri: and certification by the Japanese Plant Protection Service that fruits are free of X. citri (Goto, 1992).

Cultural Control

The disease has attracted widespread attention because of the serious efforts that have been made for eradication; these include destruction of citrus trees on a large scale an the implementation of strict international plant quarantine regulations against the pathogen (Stall and Civerolo, 1991; Goto, 1992).

The use of canker-free nursery plants is the first essential step in the management citrus canker. Windbreaks established around citrus groves reduce disease. Pruning of angular shoots which hold canker lesions removes overseasoning inocula. Periodic spraying with insecticides to control the leaf miner, Phyllocnistis citrella, reduces infection sites (Goto, 1992).

Biological Control

Interactions between X. citri and antagonistic bacteria including Bacillus subtil (Pabitra et al., 1996), Pantoea agglomerans (Goto et al., 1979), Pseudomonas syringae (Ohta, 1983) and P. fluorescens (Unnamalai and Gnanamanickam, 1984) have been reported in vitro and in vivo. However, the practical usefulness of these bacteria in controlling the pathogen has not been proved.

Chemical Control

The disease cannot be controlled by chemicals after it has reached epidemic proportions. Therefore, the prevention of primary infection on spring shoots is emphasized. this is achieved by spraying copper compounds 10-14 days after the first shoots emerge in the spring (Stall et al., 1981). Reduction of disease on spring shoots reduces inoculate for subsequent developing shoots.

Requirements and Reagents

Infected fruit or twig

1:1000 HgCl₂ solution
Sterile distilled water
Sterile empty petri plates

Sterile Potato Dextrose Agar (PDA) plates
Sterile nutrient agar slant
Sterile Biochemical media, which are required to identify the pathogen.

Sugar such as Glucose, Mannitol in peptone water base.
Nitrate reduction medium
Reagents for nitrate reduction test

Procedure

Locate affected part of the fruit showing cankerous spot. Cut a piece of the cankerous part and observe bacterial ooze under low power in wet mount preparation.
Surface sterilize cankerous part with 1:1000 HgCl solution followed by three consecutive washings in sterile distilled water.
Prepare saline suspension.

Transfer loopful of suspension o sterile PDA plate and streak inoculated for isolation.
Incubate plate at room temperature for 2-3 days.
After incubation observe isolated colonies of Xanthomonas sp. which are yellow in colour with glistening surface.

Record colony characteristics of any one isolated colony.
Inoculate saline suspension in various biochemical media.
Record the results of biochemical tests after 24 h of incubation.

Identify pathogen with the help of results of biochemical tests obtained.

Observations and Results