Biological Characteristics of the Causative Agent of Cotton

Gommosis Xanthomonas Campestris Var Malvacearum

Sh. Gulmurodova

and R. Sattarova

Tashkent State Agrarian University, 100140, University str. 2, Tashkent, Uzbekistan

Keywords: Cotton Gommosis, Xanthomonas Campestris, Pathogenic Features.

Abstract: The following article is devoted to the study of morphological-cultural and pathogenic features of six strains

of the causative agent of cotton gommosis - Xanthomonas campestris var malvacearum, isolated by us from

cotton varieties Sultan and S–6524. The study of the fermentation of carbohydrates and alcohols of six strains

of Xanthomonas campestris var malvacearum showed that all the strains we studied ferment carbohydrates

and alcohols to form acid, have the ability to break down protein and peptone to form hydrogen sulfide, and

cause diseases of cotton gommoses. The study of the morphology of Xanthomonas campestris var

malvacearum showed that the causative agent of cotton gommosis has flagella, capsule, shell and nucleoids,

and polymorphism of both the individuals and their nuclear apparatus is characteristic of diurnal cultures

1 INTRODUCTION

As known that diseases of cotton significantly reduce

its yield both in quantity and quality, worsen the

textile properties of the fiber and the quality of the

seeds sown. The threat to the harvest of raw cotton

from numerous diseases constantly exists. Gommosis

causes significant damage to cotton production.

Nowadays, cotton gommosis is registered in

almost every country where cotton is grown. This

disease is considered as a serious threat to cotton in

India, Pakistan, China, the countries of the former

Soviet Union, South America, and Australia.

Gommosis was first described by G.F. Atkinson

in 1891. The first report on the appearance of cotton

gommosis in Central Asia was made by R.R.

Schreder in 1903.

Gommosis affects the aboveground organs of

cotton cotyledons and real leaves, stipules, stems,

bracts, flowers, boxes. The signs of the disease in all

parts of the plant are basically the same. In the first

stage of the disease, the affected parts are covered

with oily round, angular or elongated spots, which

then dry out, darken, become covered with dried

mucus and die off.

https://orcid.org/0009-0008-4792-8451

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In 1901, Erwin Smith established the bacterial

origin of cotton gommosis and isolated it into a pure

culture.

The author gave the first scientific name to the

causative agent of cotton gommosis Bacterium

malvacearum. Bergey (1936) et al called Phytomonas

malvacearum. In 1949, M.A.Krasilnikov restored its

former name Pseudomonas malvacearum, noting as a

characteristic indicator of the genus Pseudomonas the

polarly located flagella of non–spore-bearing rods,

the presence of fluorescent pigment and some other

properties, not considering it advisable to isolate

phytopathogenic forms into a special genus -

Phytomonas.

V.P.Israilskiy (1960) includes the causative agent

of gommosis in the genus Xanthomonas isolated by

Stoughton, which is characterized by abundant mucus

formation, yellow color of colonies and monotrichial

flagellation.

Many scientists have studied the biological

properties of cotton gommosis (Askarova, 1960;

Babayan, 1963; Wickens, 1961; Yogan, 1960; Jones,

1983; Grigoryants et al. 2009; Bobonazarov, 2012).

X. malvacearum according to W.H. Burkholder

(1932) does not form acids on dextrose, sucrose and

lactose.

338

Gulmurodova, S. and Sattarova, R.

Biological Characteristics of the Causative Agent of Cotton Gommosis Xanthomonas Campestris Var Malvacearum.

DOI: 10.5220/0014268800004738

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 4th International Conference on Research of Agricultural and Food Technologies (I-CRAFT 2024), pages 338-344

ISBN: 978-989-758-773-3; ISSN: 3051-7710

According to Nizametdinova (1968), the

causative agent of cotton gommosis belongs to a

weak acid-forming agent.

X. malvacearum as an acid-forming agent was

described by N.A. Krasilnikov, Bergey, Israilskiy,

Nizamitdinova.

Other researchers like, Smith, Yevis, Yachevskiy,

Gorlenko think that, the causative agent of gommosis

does not form acids on environment with sugars.

Data on the morphological-cultural properties of

the causative agent of cotton gommosis is limited and

contradictory.

Therefore, the literature data indicate that many

researches have been devoted to the study of the

morphological-cultural properties of the causative

agent of cotton gommosis (Verderevsky, 1960;

Babayan, 1963; Marupova, 1968; Sattarova, 1973;

Shukri Mohamed El Gremi, 1990; Rashidov, 2003;

Fallhzaden, Ahmadzaden, 2010; Gulmurodova,

2023).

However, the analysis of the literature materials

indicates the need to continue research on the

morphological-cultural characteristics of the

pathogen causing cotton gommosisn world practice,

it has been proven that in pneumatic diluents it is most

acceptable to use landing discs rotating along a

vertically longitudinal surface, and most companies

producing pneumatic diluents produce diluents

equipped with just such discs. The research paper

considers the main parameters of the landing disc,

which rotates on a vertically longitudinal surface, that

is, on a horizontal axis located transversely to the

direction of movement of the seal (Fattah, 1976;

Babanazarov et al., 1993; Babayan, 1963;

Verderevsky, 1960; Grigoryats et al., 2009;

Beltyukova, 1968).

It is known that in order to obtain higher crop

yields, it is necessary to evenly distribute seeds

throughout the field, that is, plant them evenly to the

desired depth, ensuring a given range and distance to

the node. In world practice, when using seeds

prepared with high quality, seed sowing is used in a

clear norm, one seed per slot.

In addition to the general requirements, specific

planting requirements are developed according to the

climate and soil of each area. In particular, the soil

and climatic conditions of Uzbekistan are such that in

the spring sowing period, after precipitation, in most

cases tar appears in the soil, and seeds planted in one

grain risk getting stuck under it, failing to split the

resin.For this reason, when planting rotten seeds or

other seed materials, it is advisable to sow them in a

slot way, that is, laying 2-3 seeds in each slot

(Gulmurodova & Sattarova, 2023; Gorlenko, 1966;

Marupova, Sattarova, 1973; Rashidov et al., 2003;

Gremi, 1990).

2 MATERIALS AND METHODS

The objects of the research were samples of diseased

cotton plants of S-6524 and Sultan variety affected by

gommosis, as well as isolates of isolated by us from

diseased cotton plants.

Isolation of the causative agent of cotton

gommosis was carried out from cotyledon leaves

affected by gommosis according to the method of K.I.

Beltyukova (1968).

A section of cotton leaf tissue affected by

gommosis was placed for 2-3 minutes in 96% alcohol,

which was then thoroughly washed with sterile water

and placed in Petri dishes with potato–glucose agar.

Pure cultures of phytopathogens were obtained by

three-times re-sowing of individual colonies on solid

nutrient environment. The appearance and size of

colonies were determined on various nutrient

environment.

Cell morphology and size, Gram staining, were

studied using light microscopy.

The cultural-morphological biochemical and

physiological properties of microorganisms were

studied according to generally accepted methods and

tests described in the relevant manuals (Yegorov,

1976; Bergey S of terminative bacteriology1974).

The morphology of colonies and cells was studied

on potato agar, meat– peptone broth and Ashby

nitrogen-free medium. The shape and size of colonies

and cells were determined. The colony structure was

studied under a magnifying glass and a microscope.

The cultural properties were determined by the

nature of growth on oblique potato agar, meat peptone

broth, gelatin, potato slices.

In order to study the biochemical properties of the

studied strains, X.malvacearum was sown on a color

row containing glucose, sucrose, lactose, maltose,

levulose, xylose, arabinose, mannitol, glycerin,

dulcite, sorbitol, sallicin in an amount of 0.5%. The

results were taken into account on days 3,5,7.

Gelatin was used to identify proteolytic

properties. Sowing on gelatin was performed by

injection of a loop. The culture tubes were left at room

temperature and scanned 2-3 times a week.

The formation of ammonia was established by

reaction with the Griss reagent. Indole was

determined by the method of Morelli and Beltyukov

(1968) using strips of filter paper impregnated with a

saturated solution of oxalic acid. In the presence of

indole, the indicator paper is colored red.

Biological Characteristics of the Causative Agent of Cotton Gommosis Xanthomonas Campestris Var Malvacearum

339

Nitrate reduction was determined by the reaction

of the studied culture sown in a meat-peptone broth

containing 0.1% potassium nitrate with Griss reagent.

To identify the ability of bacteria to hydrolyze

starch, they were sown with strokes on meat –

peptone agar containing soluble flagella was detected

by staining a suspension of Xanthomonas

malvacearum with silver nitrate according to the

Nefedov method (Peshkov, 1855). The capsule was

stained using the Anthony method (Peshkov, 1955).

The capsule was colored using the Anthony

method. The smears are dried in air and stained

without fixing in the cold with a 1% solution of

crystal violet, the paint is washed off with a 2%

solution of copper sulfate, dried and washed with

water.

The coloring on nuclear elements was carried out

as follows: the smears were dried in air, fixed with

Carnois liquid for 15 minutes, placed in a wet

chamber, a ribonuclease solution was poured on top

of the smears at a concentration of 2 mg/ml and

placed in a thermostat for 1 hour at t 37

C. They were

washed off under running tap water and painted

according to Gimsa for 1 hour at 37

С.

3 RESULTS AND DISCUSSION

We have isolated six strains of Xanthomonas

campestris var malvacearum from cotton leaves of

Sultan and S-6524 cotton varieties affected by

gommosis.

The structure of X.malvacearum cells was studied

by light microscopy. The method of prints from the

surface of the agar was used for the work.

X.malvacearum has mobility, there was used the

method to clarify the nature of flagellation. As a result

of the staining of X.malvacearum to identify flagella,

it was found that Xanthomonas malvacearum has

monotrichial flagella.

There was also conducted research on staining for

nuclear elements with pretreatment with RNA – aza.

Smears prints were prepared through 2, 6, 10, 12, 14,

and 24 hours after sowing on the surface of potato

agar.

To make completely sure that the well–colored

granules and strands located in the center of

X.malvacearum cells are nucleoids and have a DNA

nature, the preparations were treated before staining

(azur – eosin) first with RNA aza and then with DNA

aza. As a result of such treatment, preparations

consisting of cells devoid of any colored structures

were obtained.

In four-hour cultures, the length of individuals

increases. In most individuals, the nucleotides are in

the stage of division. There are cells with three

nucleotides.

Starting from six-hour cultures, the

polymorphism characteristic of X. malvacearum

manifests itself, reaching its greatest development in

24 hour-cultures. Giant individuals (4-9 mmc) with

strands of nuclear matter appear in the smears from

the seals.

In the studied isolates, the cells of Xanthomonas

campestris var malvacearum are small in shape,

slightly tapering towards the end, movable rods 1.5

mk long, 0.5 mk wide, with rounded ends, nonporous,

gram-negative, forming a capsule. Some strains have

cells 9 microns long in 18-hour or daily cultures.. The

presence of long chains of cells indicates a significant

polymorphism of the microorganism.

The optimal temperature for growing X.

malvacearum culture in laboratory conditions is 20-

С. The morphology of young cells (6-10 hour)

cultures varies when growing on different nutrient

media.

On potato agar, the culture of Xanthomonas

campestris var malvacearum has the appearance of

slender sticks. On meat–peptone agar, the cell

contours are vague, indistinct, the cells are irregularly

shaped, short and strongly thickened. On Ashby's

nitrogen-free environment, the sticks are large and

slender. The measurement of bacteria grown on

various media gave the following results: on meat–

peptone agar -1,9-2,5/0, 8-1,0, 8-1,0 mk, on potato

1,25 – 1,5 /0,5 – 0,8 mk, on pepton – 1,3 -1,5/0,9 –

1,0 mk, on Ashby – 1,8 /0.3 – 1 mk.

On sterile potato slices with a small amount of

water, the growth of X.malvacearum is moistly

convex, shiny, pale yellow, waxy yellow with growth,

a brown plaque forms, the potatoes darken.

The growth on oblique potato agar is good,

strong, spreading, convex, the growth surface is

smooth, moist, the edges are even, the pigment is

yellow.

The development of X.malvacearum cultures on

meat-peptone broth causes uniform turbidity of the

medium. At the bottom of a small slimy sediment,

turbidity rises when shaken. A loose, granular film

forms on the surface of the meat-peptone broth.

The morphology of colonies of X.malvacearum

was also studied on various media. On potato agar in

Petri dishes, X.malvacearum bacteria form round,

flat, soft, pale yellow colonies with smooth edges,

which darken on 5-7 days. The diameter of adult

colonies is 1-10 mm. In transmitted light, colonies

have a homogeneous structure, sometimes

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concentric, radial, and spindle-shaped. On days 7-10,

a seal forms in the center of the colonies. On meat-

peptone agar, colonies are rounded, pale yellow,

darken with age. On the Ashby mineral medium, the

colonies are small, transparent, shiny, the edges of the

colonies are smooth in the transmitted light and have

a granular structure.

When all strains of X.malvacearum were sieved

on potato agar to obtain individual colonies, 4 types

of colonies were found: 1. Smooth, round, shiny,

convex colonies, yellowish–green, with smooth

edges, in the transmitted light there is a seal in the

center; 2. Slightly convex, shiny, slimy, greenish–

yellow, the edges are smooth, transparent, concentric

circles are visible in the transmitted light; 3. Large,

flat, shiny grayish-yellow, the edges are transparent,

structures in the form of grains are visible in passing

light, which are located radically along the edge and

acquire a fusiform shape; 4. Convex, round, slimy,

bright yellow, smooth edges, homogeneous in

passing light.

Type I and II colonies are most characteristic of

the causative agent of cotton gommosis Xanthomonas

campestris var malvacearum and contains 80%.

At this point, the morphological properties of the

colonies were preserved, and no further dissociation

was observed. According to the literature,

Xanthomonas malvacearum forms acid on sugars,

peptonizes and coagulates milk, and hydrolyzes

starch (Gorlenko, 1961; Babayan, 1963).

The study of the fermentation of some

carbohydrates and alcohols showed that the studied

strains of Xanthomonas malvacearum slightly differ

in their ability to ferment individual carbohydrates

from the control strain. As a control strain, we used

the data given in the determinant by N.A.Krasilnikov,

Bergey.

The control strain forms acid without gas on

glucose, sucrose, lactose, galactose, xylose, raffinose,

glycerin, does not ferment arabinose, rhamnose,

dulcite and mannitol, coagulates and peptonizes milk,

hydrolyzes starch, indole and hydrogen sulfide does

not form.

The studied strains of Xanthomonas

malvacearum, with the exception of strains 2 and 5,

do not form acid on xylose. Only strains 1 and 3 form

acid on maltose, which brings them closer to the

control one. Strain 3 does not ferment sucrose (Table

1).

As a result of studying the proteolytic activity, it

was found that the studied strains of Xanthomonas

malvacearum have the ability to cleave protein and

peptone with the release of hydrogen sulfide (Table

2), and some strains (1, 2, 3) intensively secrete

hydrogen sulfide, others (5, 6) have a weak reaction

to the formation of indole in all strains of

Xanthomonas campestris var malvacearum is

negative.

Due to N.A. Krasilnikov, M.V. Gorlenko, V.P.

Israelsky, the causative agent of cotton gommosis

does not emit hydrogen sulfide and indole.

Xanthomonas malvacearum cultures slowly

dilute the gelatin, which remains transparent. Strains

1, 2, 3 dilute gelatins more strongly than strains 5 and

6 (tab 2).

All researched strains of Xanthomonas

malvacearum have proteolytic activity and

saccharolytic properties. Xanthomonas malvacearum

strain peptonizes and coagulates milk with the

exception of the fifth strain, which only peptonizes

milk. The rate and intensity of hydrolysis in all strains

of Xanthomonas malvacearum is different; strains (1,

2, 3) hydrolyzed starch already on the 3rd day, and (5

and 6) – on the 7th day.

In order to identify reducing properties, a litmus

serum was used, on which cultures develop in

different ways. Some strains (1, 2, 3, 4 and 5) alkalize

the medium, others (2 and 6) do not change it (tab 2).

Microorganisms are able to restore nitrates.

However, according to N.A.Krasilnikov (1949),

Xanthomonas malvacearum does not restore nitrates.

According to the data, only strains 2 and 4 are capable

of reducing nitrates to nitrites.

In Clark’s medium, no strain of Xanthomonas

malvacearum secretes acetyl–methyl–carbinol.

On the Klodnisky–Peshkov medium, all strains

grew throughout the entire thickness of the medium.

Consequently, Xanthomonas malvacearum is a

facultative anaerobe.

The studied strains of Xanthomonas

malvacearum do not hydrolyze fats, i.e. they are not

capable of forming lipase.

Cultures obtained from different types of colonies

differ in cultural and physiological properties.

Strains differing in colony morphology differ

from the main strains in some cultural-physiological

features (Table 3).

In cultures isolated from colonies of types II and

IV (strains 3/II, 3 IV), proteolytic activity is more

pronounced than in the main strain 3.

In cultures isolated from colonies of types I and

III (strains 2 I and 2 III), proteolytic activity turned

out to be weaker than that of the main strain 2. In

addition to this, strains 2 (I) and 2 (III), unlike the

main one, do not dilute gelatin and do not form acid

on xylose.

Biological Characteristics of the Causative Agent of Cotton Gommosis Xanthomonas Campestris Var Malvacearum

341

Table 1: Fermentation of carbon sources by various strains of Xanthomonas campestris var malvacearum.

Strains Omelyanskiy medium

Glucos

Sucros

Lactos

Maltos

Levulos

Xylos

Mannit

Glyceri

Arabinos

Dulcit

Sorbit

Salici

1 + + + + + - - + - - - -

2 + + + - + + - + - - - -

3 + - + + + - - + - - - -

4 + + + - + - - + - - - -

5 + + + - + + - + - - - -

6 + + + - + + - + - - - -

Contr

+ + + + + + - + + - - -

Note: + existence of acid, - absence of acid

Table 2: Reducing and proteolytic properties of Xanthomonas campestris var malvacearum.n

Strains

Reducing properties Proteolytic properties

Nitrate recovery

Reduction of

litmus serum

Release of

hydrogen sulfide

Dilution of

gelatin

Starch

hydrolysis

1 + AF ++++ +++ +++

2 - AF ++++ +++ +++

3 - AF ++++ +++ +++

4 + AF +++ ++ ++

5 - AF ++ + +

6 - AF ++ ++ ++

Control N/A N/A - ++ ++

Note

AF - change of the medium towards alkali formation

+ - intensity of proteolytic properties, starch hydrolysis

Table 3: Cultural-biochemical properties of colonies differing in morphology Xanthomonas malvacearum.

Strains Omelyanskiy medium

Meat

eptone

roth

Litmus

serum

Mil

Gelatin

Hydrogen

sulfide

Glucose

Sucrose

Lactose

Maltose

Mannitol

Dulcite

Sorbitol

Mannose

Xylose

Glycerin

+ - + + - - - + - -

Wall film

mud

sludge

AF Peptonizes ++ +

3 (II)

+ - + - - - - + - -

Small film

of mud

AF Peptonizes +++ ++

3 (IV)

+ - + - - - - + - -

Small film

of mud

Peptonizes and

coagulates

+++ +++

+ + + - - - - + - -

Small film

of mud

- Peptonizes ++ +++

2 (I)

+ + + - - - - + - -

Muddy

film

Coagulates,

peptonizes

- +

2 (III)

+ + + + - - - + - -

Mud is a

small film

Peptonizes and

coagulates

- +

Note: AF - alkali formation + - presence of hydrogen sulfide acid, reduction of nitrates, dilution of gelatin.

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The main criterion for the belonging of unknown

crops to the species Xanthomonas campestris var

malvacearum is their pathogenicity to cotton.

Regarding to this, we studied the ability of

Xanthomonas malvacearum strains isolated by us to

cause the incidence of cotton gommosis. For this

purpose, artificial contamination of Sultan and S-

6524 cotton seeds was carried out.

The results of artificial infection of cotton seeds

with the studied strains of X. malvacearum showed

that all strains cause cotton disease with gommosis,

but not to the same extent (Peshkov, 1955; Vinay,

2013; Fallahzadeh & Ahmadzaden, 2010; Eholvel &

Kurundkar, 2007; Yogan, 1960; Hillocks, 1992;

Jnnes, 1992; Wickens, 1961; Saidova et. al., 2023).

The disease occurred after seven days, from the

beginning of the experiment. The clearest results

were observed in 15-day-old seedlings.

Strains 1, 2, 3 strongly affected Sultan cotton. The

cotyledon leaves were completely covered with large

oily spots.

Strains 5 and 6 were weakly affected. 2-3 small

oily spots appeared on cotyledon leaves.

The difference in the degree of pathogenicity was

also manifested in varieties S-6524.

Strains 1, 2, 3 affected the studied varieties more

strongly, more intensively, and 5, 6 weaker.

Consequently, all the studied strains isolated from

cotton leaves belong to the species X. malvacearum,

because with artificial infection of seeds, symptoms

of cotton gommosis appeared on cotyledon leaves.

4 CONCLUSIONS

Based on the experiments of the conducted studies, it

can be concluded that all cultures isolated from cotton

leaves affected by gommosis are representatives of

the species Xanthomonas campestris var.

malvacearum, since they are similar to the species in

terms of the main cultural morphological,

physiological and pathogenic signs. Xanthomonas

malvacearum described in the determinants of

Bergey and N.A. Krasilnikov. While studying the

cytology of Xanthomonas malvacearum, we found at

the level of light microscopy that Xanthomonas

malvacearum has flagella, capsule and isolated

nucleoids.

X. malvacearum cells aged 18-24 hours are

characterized by the presence of polymorphism, both

of the individuals themselves and their nucleoids.

Strains differing in morphology of the colony differ

from the main strains in some cultural-morphological

features. It’s worthy to note that some of the

differences we have established between X.

malvacearum strains may be strain-specific features

rather than species-specific. To identify the causative

agent of cotton gommosis, we need to conduct

additional studies at the molecular level. Research in

this direction will continue

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