XANTHOMONAS CAMPESTRIS: DESCRIPTION, CULTIVATION AND PATHOLOGIES - BIOLOGY - 2025

Xanthomonas campestris is a species of proteobacteria (class: Gamma Proteobacteria, order: Xantomonadales, family: Xanthomonadaceae) phytopathogenic that affects important crops.

X. campestris presents an epiphytic phase on the plant in which it does not harm it. This phase is prior to infection, it is generated when the bacteria proliferate due to favorable environmental variations. Infection by this species causes a variety of symptoms in the infected plant that can eventually degenerate into death.

Figure 1. Symptoms caused by infection with X. campestris. Source: author Ninjatacoshell, Wikimedia Commons.

X. campestris is also well known for producing the biopolymer xanthan gum or xanthan, a polysaccharide that excretes into the medium (exopolysaccharide) and increases the viscosity of aqueous solutions.

The exopolysaccharide xanthan was the first commercially important byproduct generated by the fermentation processes of corn starches. It is currently produced in large quantities and has many applications due to its characteristics as a thickener and emulsifier. Xanthan is used in the food, pharmaceutical, cosmetic, agricultural, and oil industries, among others.

Description

Xanthomonas campestris is a gram-negative, obligate aerobic, facultative saprophyte bacillus. It is mobile, between 0.2 and 0.6 µm wide, and between 0.8 and 2.9 µm long. It can appear as a solitary individual or forming filaments, surrounded by xanthan, the exopolysaccharide they produce.

Xanthan favors the formation of X. campestris biofilms and also exerts a protective action on the communities established in this structure, when sudden changes in temperature, pH, ultraviolet radiation, marked osmotic variations and / or humidity decrease occur.

Interaction with the plant

This species has several mechanisms to evade the defense responses of the plants it infects. The first barrier of the plant against a bacterial infection is the cell wall and surface substances with antimicrobial activity.

X. campestris can infect the plant through its foliar stomata (pores where gas exchange occurs with the environment), its hydatodes (a type of stoma through which excess water exudes), or through existing wounds.

Plants generally close their stomata when attacked by microorganisms. However, X. campestris produces a virulence factor that prevents the stomata from closing, thus favoring the entry of more bacteria into the plant from the external environment.

When the bacteria are inside the plant, they prevent the transport of water, by obstructing the vascular tissues. The result is necrosis of the leaves and wilting of the infected parts.

In addition, X. campestris produces a compound called neutral cyclic β- (1,2) glucan that prevents the expression of defense genes in the plant. These compounds can be associated with the bacterial periplasmic space or can be excreted to the extracellular environment, favoring the mobility of the bacteria, its virulence and the formation of biofilms.

Xanthan

The xanthan produced by the Xanthomonas acts as a virulence factor, suppressing the immune response of the infected plant and increasing the infectivity of the bacteria.

Xanthan is a polysaccharide made up of units of 5 sugars that are repeated (2 glucoses, 2 mannoses and 1 glucuronic acid) and polymerize.

The synthesis of xanthan depends on an operon called cluster gum (a set of genes that make up a functional unit), which has 12 genes that are under the control of a single promoter region.

Isolation of

X. campestris pv. campestris can be isolated from leaf tissue that has "V" -shaped spots or from damaged vascular tissue, or the neck of the plant, that is, from the injured areas of the plant.

To obtain strains of X. campestris, the injured area is selected as a sample (leaf or fruit spots or cankers). If no injury is observed in the plant, the tissue most susceptible to being damaged is taken as a sample, and they are analyzed by culture media and by the polymerase chain reaction (PCR) technique.

Culture media

Among the culture media used are the following:

Milk tween

For an initial isolation of microorganisms from samples of plant tissue, Milk Tween (MT) medium can be applied:

10 ml of skim milk, 0.25 g of CaCl ₂, 10 g of peptone protease No. 3, 15 g of Bacto agar, 0.5 g of tyrosine, 10 ml of Tween 80, 80 mg of cephalexin (in 2 ml of NaOH at 4%), 200 g of cycloheximide (in 2 ml of 75% methanol), 100 mg of vancomycin (in 1 ml of distilled water).

Skim milk, cephalexin, cycloheximide, and vancomycin solutions must be sterilized by filtration and added to the medium at 50 ° C.

King's B

After allowing the bacterial colonies to grow in the MT, the ones most similar to X. campestris (yellow pigmentation colonies at 72 and 120 hours of culture) can be transferred to King's B medium:

20 g of peptone protease No.3, 20 g of agar agar, 1.5 g K ₂ HPO _4, 1.5 g MgSO ₄ x / H ₂ O, 10 ml of glycerol, 700 of distilled water.

The medium should be heated to 80 ° C with stirring, made up to 1 L with distilled water and homogenized, and the pH should be adjusted to 7.2. Sterilize at 121 ° C for 15 minutes.

The rich culture medium PYM or YMM has also been used in the culture of X. campestris.

Figure 2. Culture of Xanthomonas in solid medium. Source: Jarober3, via Wikimedia Commons

PYM

To prepare the PYM, for every 1000 ml of total volume, add: 10 g of glucose, 5 g of peptone extract, 3 g of malt extract and 3 g of yeast.

If you want to grow in solid medium in Petri dishes, then 15 g of agar should also be added to the mixture.

Ymm

To prepare the YMM medium, you need for each 1000 ml of total volume: 10 g of glucose, 1 ml of MgSO ₄: 7H ₂ O solution (10 g / L), 1 ml of a CaCl ₂ solution (22 g / L), 1 ml of a K ₂ HPO ₄ solution (22 gr / L), 1 ml of a FeCl ₃ solution in 0.1 M HCl (2 g / L), casamino acids 0.3% m / v (amino acids from hydrolysis of casein) and 11% v / v of sodium glutamate solution.

Incubation conditions

The incubation conditions of the bacterial strains of X. campestris should be 27 or 28 ° C, and in the case of liquid culture media, continuous stirring should be maintained at 200 revolutions per minute (rpm).

Xanthan production

If the production of xanthan in a fermentation process is desired, glucose, sucrose, or corn syrup (between 20 and 40 g / L), among other nutrients that provide nitrogen, must be supplied as a carbon source.

Detection of metabolic activity

To detect the presence of viable X. campestris in plant tissue, some researchers recommend measuring metabolic activity, rather than microbial growth in laboratory culture.

The measurement of metabolic activity has been carried out using a viability indicator through the electron transport system. This compound is called tetrazolium and its salts accept electrons from hydrogen, generating formazan, a substance insoluble in water. Thus, the appearance in the middle of formazan is an indicator of cellular metabolic activity.

One of the X. campestris culture media to perform this viability test contains tetrazolium chloride (TTC), triphenyl tetrazolium chloride and other additives such as sodium chloride and sugars. It is a medium with the following substances for a total volume of 500 ml: 5 g of peptone, 0.5 g of hydrolyzed casein, 2.5 g of glucose and 8.5 g of agar.

Pathophysiology

The bacterium X. campestris is the causal agent of numerous diseases that affect the leaves of ornamental plants (such as Anthurium andreanum) and common beans (Phaseolus vulgaris L.). They also affect the fruits of stone fruit, such as almond, nectarine, cherry, peach, apricot, plum, among others.

X. campestris is known to affect the Brassicaceae or cruciferous family, being among the 10 phytopathogenic species most dangerous for agricultural activity, particularly in the tropics.

For example, X. campestris causes black rot disease in cauliflower (Brassica oleracea), broccoli (B. napus), Chinese cabbage (B. pekinensis), turnip (B. rapa), mustard (B nigra), radish (Rhaphanus sativus) and cabbage (B. fruticulosa).

Figure 3. Leaf affected by X. campestris. Source: David B. Langston, via Wikimedia Commons

The symptoms produced by X. campestris appear initially on leaves and then may appear on fruits and branches. They involve irregular and angular yellowish leaf spots (1 to 5 mm in diameter) limited by the veins that are eventually necrotized.

Foliar burns also occur; spots on the fruits; vascular wilt and the appearance of chlorotic or necrotic lesions in the shape of a "V".

The spots appear on the leaf edges and surrounding its midrib. Leaf loss may occur on the plant. Green spots appear on the fruits that become necrotic, and may also crack them. Cankers can also occur.

References

1. Dow, JM, Crossman, L., Findlay, K., He, Y.-Q., Feng, J.-X., & Tang, J.-L. (2003). Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proceedings of the National Academy of Sciences, 100 (19), 10995–11000. doi: 10.1073 / pnas.1833360100
2. Hayward, AC, Swings, JG and Civerolo, EL (1993). Xanthomonas. Springer Netherlands. pp 407.
3. Papagianni, M., Psomas, S., Batsilas, L., Paras, S., Kyriakidis, D. and Liakopoulou-Kyriakides, M. (2001). Xanthan production by Xanthomonas campestris in batch cultures. Process Biochemistry, 37 (1), 73-80. doi: 10.1016 / s0032-9592 (01) 00174-1
4. Rosalam, S., & England, R. (2006). Review of xanthan gum production from unmodified starches by Xanthomonas campestris Enzyme and Microbial Technology, 39 (2), 197–207. doi: 10.1016 / j.enzmictec.2005.10.019
5. Stewart, P. and Globig, S. (2011). Phytopathology in plants. Apple Academic Press. pp 334.