MICROBIAL EXAMINATION OF FOOD- Viable colony count, Examination of fecal Streptococci

 

Microbiological examination of a food is required in order to estimate its shelf-life or its suitability for human consumption. The total viable count or more often, the numbers of a particular component of the total flora such as moulds in a cereal, psychrotrophic bacteria in a product to be stored at low temperature, anaerobes in a vacuum- packed food, or yeasts in a fruit beverage may be needed. Or it may be required to ensure that a food meets established microbiological criteria.

The total mesophilic plate count is widely used as an indication of the microbiological quality of foods unless they are known to contain large numbers of bacteria as a result of their preparation such as fermented milk and meat products.

Microbiological analysis is also required in case of spoilage or foodborne illness where the presence of a pathogen is implicated. The methods for determining an estimate of the total mesophilic count are different from those for identifying the presence of a pathogen, or its isolation for further study.

The isolation of specific pathogens, which may be present in very low but significant numbers in the presence of larger numbers of other organisms, often requires quite elaborate procedures. It may involve enrichment in media which encourage growth of the pathogen while suppressing the growth of the accompanying flora, followed by isolation on selective diagnostic media, and finally the confirmatory tests. Microbiological criteria or the investigation of an outbreak of foodborne illness thus require the monitoring of certain products for specific pathogens, but the difficulties in detecting low numbers of pathogens make it impracticable as a routine procedure.

Indicator organism

An alternative to monitoring for specific pathogens is to look for an associated organism present in much larger numbers – an indicator organism. This is a concept developed originally for pathogens spread by the faecal-oral route in water. A good indicator organism should always be present when the pathogen may be present. It should be present in relatively large numbers to facilitate its detection. It should not proliferate in the environment being monitored and its survival should be similar to that of the pathogen for which it is to be used as an indicator.

Escherichia coli is a natural component of the human gut flora and its presence in the environment, or in foods, generally implies some history of contamination of faecal origin. In water microbiology E. coli meets these criteria very well and is a useful indicator organism of faecal pollution of water which may be used for drinking or in the preparation of foods.

There are, however, limitations to its use in foods where there appears to be little or no correlation between the presence of E. coli and pathogens such as Salmonella in meat, for example. E. coli cannot may not grow in water but, it can grow in the richer environment provided by many foods.

            Thus, routine microbial examination of food mainly relies on viable colony count, and examination of fecal streptococci.

v Viable colony count

Viable colony count/Plate count is used to estimate the number of viable cells that are present in a sample. The most common procedure for the enumeration of bacteria is the viable plate count.

*       It relies on bacteria growing a colony on a nutrient medium. A viable cell count allows one to identify the number of actively growing/dividing cells in a sample.

*       In this method, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium. The suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then incubated under conditions that permit microbial reproduction.

*       It is assumed that each bacterial colony arises from an individual cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor, the number of bacteria in the original sample can be determined.

Drawbacks

*       Traditional plate counts are expensive in Petri dishes and agar media

*       The major disadvantage is that it is selective and therefore biased.

*       The nature of the growth conditions, including the composition and pH of the medium used as well as the conditions such as temperature, determines which bacteria in a mixed population can grow.

*        Since there is no universal set of conditions that permits the growth of all microorganisms, it is impossible to enumerate all microorganisms by viable plating.

Key points

*       Selective procedures for the enumeration of coliforms and other groups can be designed using selective media/conditions.

*       For routine laboratory investigations, it is a good and sensitive method of detecting the presence of a viable bacterium.

*       This is the basis of the traditional pour plate, spread plate or Miles and Misra drop plate and is widely used in microbiology laboratories.

*        In the pour plate method, a sample (usually 1 ml) is pipetted directly into a sterile Petri dish and mixed with an appropriate volume of molten agar.

*       Even if the molten agar is carefully tempered at 40 – 45^oC, the thermal shock to psychrotrophs may result in them not producing a visible colony.

*       The spread-plate count avoids this problem and also ensures an aerobic environment but the sample size is usually limited to 0.1 ml

 

*       Since we usually have no idea of how many bacteria are in a sample, it is almost always necessary to prepare a dilution series to ensure that we obtain a dilution containing a reasonable number of bacteria to count.

*        Dilutions in the range 10^-1(1/10) to 10^-8 (1/100,000,000) are generally used, although with particular types of samples the range of dilutions can be restricted.

^*             For example, for water that is not turbid, the dilution needed can be 10^{− 6}

The Miles and Misra drop count and spiral plater have been developed to reduce costs of traditional plating methods.

v Miles–Misra technique

The Miles and Misra Method (or surface viable count) is used to determine the number of colony forming units in a bacterial suspension or sample. The technique was first described in 1938 by Miles, Misra and Irwin.

*       The inoculum / suspension is serially diluted. A calibrated dropping pipette, or automatic pipette, delivers drops of 20μl on petri dishes containing nutrient agar or other appropriate medium.

*       Three plates are needed for each dilution series- an average of at least 3 counts are needed.

*       The surface of the plates need to be sufficiently dry to allow a 20μl drop to be absorbed in 15–20 minutes.

*       Plates are divided into equal sectors (it is possible to use up to 8 per plate). The sectors are labelled with the dilutions.

*       In each sector, 20 μl of the appropriate dilution is dropped onto the surface of the agar (without touching the surface of the agar with the pipette) and the drop allowed to spread naturally.

*       The plates are allowed to dry before inversion and incubation at 37 °C for 18 – 24 hours (or appropriate incubation conditions considering the organism and agar used).

*       Each sector is observed for growth, high concentrations will give a confluent growth over the area of the drop, or a large number of small/merged colonies.

*       Colonies are counted in the sector where the highest number of full-size discrete colonies can be seen (usually sectors containing between 2-20 colonies are counted).

*       The following equation is used to calculate the number of colony forming units (CFU) per ml from the original aliquot / sample

Some disadvantages are that the rate of absorption of drops onto the surface of agar depends upon the environmental conditions like temperature and humidity. And, a skilled microbiologist is needed to perform serial dilution and to follow aseptic techniques.

 

v Spiral plater

A method for determining the number of bacteria in a solution by the use of a machine which deposits a known volume of sample on a rotating agar plate in a decreasing amount in the form of an Archimedes spiral. After the sample is incubated, different colony densities are apparent on the surface of the plate. By counting an appropriate area of the plate, the number of bacteria in the sample is estimated.

This method was compared to the pour plate procedure and the spiral plate method gave counts that were higher than counts obtained by the pour plate method. The time and materials required for this method are substantially less than those required for the conventional aerobic pour plate procedure.

 
 *       The spiral plater has a mechanical system which dispenses 50 ml of a liquid sample as a spiral track on the surface of an agar plate-known volume of sample is inoculated, from the center to the periphery of the plate.

*       The system is designed so that most of the sample is deposited near the centre of the plate with a decreasing volume applied towards the edge. There is a progressive decrease in the concentration of the sample.

*       This produces an effect equivalent to a dilution of the sample by a factor of 10³ on a single plate, thus saving on materials as well as saving the time required in preparing and plating extra dilutions.

Comparison of raw and analysed spiral plate images

Spiral platers are used extensively in food safety testing, as well as the pharmaceutical industry. The spiral plating method can save time and resources by cutting down on additional dilution and plating steps.

The system is not suitable for all food samples, as particulate material can block the hollow stylus through which the sample is applied.

 Advantages of spiral plating

• Reduces the number of serial dilutions needed for microbiological testing-3 dilutions obtained in 1 plate
• Reduces the number of Petri dishes needed-fewer the number of dilutions, fewer Petri dishes are required.
• The Spiral distribution enables a reduction of the counting procedure. In most cases, the count is only needed to be performed on one portion of the agar.

 

Fecal Streptococci

The fecal streptococci and enterococci are members of the genera Streptococcus and Enterococcus.  These bacteria are spherical, gram positive, grow in chains, are facultative anaerobic and ferment sugars.  The enterococci and many fecal streptococci are normal flora of the gastrointestinal tract, where they are present at high concentrations.  However, they can sometimes spread from the gastrointestinal tract and cause bacteremia, wound infections, urinary tract infections and endocarditis, more likely in new borns and in the elderly. 

Enterococci are now a concern in nosocomial infections as well as infections of non-hospitalized populations because of growing antibiotic resistance, especially to antibiotics such as vancomycin.

The streptococci and enterococci are relatively fastidious in their growth requirements because they have lost the ability to synthesize many essential nutrients. Therefore, they require several amino acids and the B vitamins to be grown in the laboratory.

Faecal streptococci are recovered from faeces in significant numbers. These species are S. faecalis, S. faecium, S. avium, S. gallinarum, S. bovis, and S. equinus. In the water microbiology, fecal streptococci are indicators of recent fecal water contamination.

*       S. bovis and S. equinus are predominately found in animals

*       S. faecalis and S. faecium are more specific to the human gut.

Streptococci can be classified on the basis of Lancefield (carbohydrate of the cell wall) antigens (A, B, C, D, E-T). Lancefield divided the streptococci into serological groups based on the presence/absence of certain carbohydrate antigens. Lancefield's Group D possesses the antigen glycerol teichoic acid.

Streptococci that posses the Group D antigen are those generally considered to be faecal streptococci—S. faecalis, S. faecium, S. avium, S. gallinarium, S. bovis, and S. equinus.

The fecal streptococci and enterococci belong to serogroup D. 

Enterococci

The enterococci include S. faecalis, S. faecium, S. durans and related biotypes. Enterococci

• grow at both 10°C and 45°C;

• survive at 60°C for at least 30 min;

• grow at pH 9.6;

• grow in the presence of 6.5% sodium chloride;

• reduce 0.1% methylene blue in milk.

They also

• exhibit resistance to the antibiotic vancomycin;

• react with streptococcal Group D antiserum;

• are bile-esculin positive;

Faecal streptococci along with total and faecal coliforms, are generally regarded as the most useful indicators of faecal pollution, and their use has been recommended internationally.

The enterococcus group (a subgroup of the faecal streptococcus that includes Enterococcus faecalis, Ent. faecium, Ent. avium and Ent. gallinarum) is a valuable bacterial indicator for determining the extent of faecal contamination of recreational surface waters. A direct relationship between swimming-associated gastroenteritis and enterococci has been established through epidemiological studies at marine freshwater bathing beaches

Detection of Faecal Streptococci

Methods for enumeration of fecal streptococci

*       Most probable number (MPN) method

*        Membrane filtration

*       Plate count methods

*       Fluorescent antibody techniques

*       Biochemical species identification systems

*       DNA-based identification systems

The methods most commonly used to assess faecal streptococci in water are the most probable number (MPN) and the membrane Filltration (MF) techniques. The MPN technique is applicable primarily to raw and chlorinated waste water and sediments, and it can be used for fresh and marine waters. The disadvantages of the MPN technique are that it requires both time and materials, requiring low-cost labour. The MF method may also be used for fresh and saline samples, but it is unsuitable for highly turbid waters. Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filltration. Moreover, suspended materials cause spreading colonies that may interfere with target colonies and thereby prevent accurate counting.

The pour plate technique (PP), is more suitable for enumerating faecal streptococci densities in polluted waters. The pour plate method is a simple technique familiar to water bacteriologists; it is less time-consuming than MPN counts and cheaper, but it is as accurate as membrane filter counts.

Mediums for the growth of fecal streptococci used in a multiple tube (most probable number; MPN) method, with a membrane filter (MIF) procedure, or by the agar pour plate technique are

*       Azide dextrose broth

*       SF medium (Streptococcus Faecalis medium)

*       BAGG broth - Buffered Azide Glucose Glycerol Medium

*       Ethyl violet azide broth/ (EVA) broth

*       KF streptococcal medium (Kenner faecal) streptococcus broth

 

The World Health Organisation recommends the use of Enterococcus agar for use with pour plates, spread plates, or membrane filtration techniques for enumerating faecal streptococci and incubation at 44°C for 48 h.

 

The MPN or multiple tube dilution (MTD) method can be used to enumerate faecal streptococci .

1.1  Presumptive isolation in azide dextrose broth (AD)

*       The AD-EVA procedure involves inoculation of dilutions of sample into replicate tubes of AD broth.

*        Positive tubes exhibit turbidity following 24-48 h incubation at 35-37°C.

*       These tubes are confirmed by transfer to Ethyl violet azide broth/ (EVA) broth, where a positive reaction is indicated by the formation of a purple "button", or dense turbidity, after 48 h incubation at 35-37°C.

*       The faecal streptococcus count is estimated from the positive EVA tubes using an MPN table.

*       Various other broths have been recommended for the recovery of faecal streptococci, including Kenner faecal streptococcus (KF) broth and Pfizer selective enterococcus (PSE) broth

*       However, the AD-EVA method is probably the most commonly used liquid medium system, and is recommended by WHO.

1.2  Presumptive isolation in AD broth for 24-48 h is followed by confirmation by streaking a portion of growth to plates of PSE (Pfizer selective enterococcus) agar that are then incubated for 24 h at 35°C.

 

2. Membrane filtration involves drawing a known volume of liquid sample through a membrane filter with a 0.45 um pore size.

*       Bacteria present in the liquid are retained on the filter surface and the filter is then transferred to a selective medium for colony development.

*       The number of colonies counted on the membrane is taken to be equivalent to the number of streptococcal cells present per volume of sample.

*        Compared to the most probable number method, membrane filtration produces results more quickly, allows a larger volume of sample to be tested, allows colonies to be counted directly, and allows the immediate biochemical testing of isolated colonies.

*       However, filtration may not be used for highly turbid samples and is not recommended for chlorinated effluent.

*       KF broth/agar (Kenner faecal streptococcus) was probably the most commonly used faecal streptococci membrane filtration medium.

*        Faecal streptococci appear on this medium as pink to dark red colonies after incubation at 35°C for 48 h.

*    When the membrane is overlaid with PSE medium, faecal streptococci colonies can be distinguished by the presence of dark brown halos indicative of esculin hydrolysis.

 

3.  The bile-esculin test is a biochemical test performed to differentiate Enterococci and group D Streptococci from non-group D viridans group Streptococci on the basis of their ability to hydrolyze esculin.

• Esculin is a glycosidic fluorescent compound, and its hydrolysis can also be observed by the loss of the fluorescence.
• The blackening of the medium and  loss of the fluorescence demonstrates a positive test. The lack of color change demonstrates a negative tube test.

·  Many organisms are capable of hydrolyzing esculin, but only a few of them can do so in the presence of bile (4% bile salts or 40% bile.). This property is utilized to identify organisms of Enterobacteriaceae, genera Streptococcus and Listeria etc.

·  The Bile-esculin test is performed on a selective differential agar; bile esculin agar, which consists of bile as well as esculin.

·  The agar contains different bile salts that inhibit the growth of other Gram-positive organisms and allows the selective isolation of Enterococci and Group D Streptococci.

·  Nowadays, bile-esculin disks are available that are commonly used for the rapid differentiation between Group D Streptococci and non-Group D Streptococci.

 

Fungal Plant Diseases- Red rot of sugarcane, Early blight of potato

 Red rot:

Organism: Glomerella tucumanensis/ Colletotrichum falcatum

Red rot disease is caused by the fungus Glomerella tucumanensis, previously called Colletotrichum falcatum. This is one of the most severe of the known diseases of sugarcane. It is widely distributed throughout the sugar­cane-growing countries of the world.

 

Symptoms

• Infection in the stem is internal and the presence of the disease is not visible externally.
• Upon splitting a diseased cane during the early stages of the disease, it will be found that the fibro-vascular bundles near the base are reddish in colour. A gummy dark-red material oozes out of the cells filling the in­tercellular spaces. The soluble pigment present in this ooze, is absorbed by the cell wall producing the characteristic red rot appearance.

·       As the disease advances the red colour spreads to the surrounding tissues and irregular discoloured blotches are formed, which may be reddish or yellowish or white with red margins.

• When the infection becomes old, the blood-red colour changes to straw colour. The stem shrinks and split cane gives sour smell and shows red tissue with white cross-bands.
• Sometimes, the pith inside the cane is filled with blackish brown liquid and exhibits alcohol odour.
• About this time the upper leaves of the stem turn pale and gradually droop down. Ultimately the entire plant withers and droops down. The first external evidences of disease are the drooping, withering, and finally yellowing of the upper leaves, followed by wilting of the entire leaves, and finally the entire plant.
• The external symptoms appear only after16 - 21 days after infection and entire cane dries off in  10 days time.

 

In areas where the disease appears in a severe epidemic form, the en­tire crop withers and droops resulting in a complete loss of crop. Though the fungus attacks all parts of the host above ground, stems and midribs of leaves are more susceptible to fungal attack.

Reddish lesion on leaf midrib  Orange to yellow leaf         Inner reddish region

Management Removal of the affected plants at an early stage and soil drenching with Carbendazim The cut ends and entire plant should be dipped in a fungicide solution (Bordeaux mixture) If the disease is noticed in the field, the leaves and canes should be collected and destroyed by burning. Treatment with Carbendazim before planting should be done Use fungitoxic chemicals like Bavistan, Benomyl, Topsin and Aretan for complete elimination of rot infection. Plant healthy crops in a disease- free area. The red rot affected field must be rotated with rice for one season and other crops for two seasons. Growing of recommended resistant and moderately resistant varieties

Red rot of sugarcane is hard to control because the stalk from which seeds are prepared has been largely affected from the time of planting, and fungicides cannot reach the infected tissues inside a diseased seed sett. Therefore careful selec­tion of red rot-free seed setts is recommended for planting. Seed should always be taken from disease-free nurseries and examined regularly. Before planting, it should be carefully examined and those which show reddening should be discarded.

 

The spread of the red rot can be prevented during the growing season by timely inspection and burning of the affected clumps followed by sanitation. Hot water treatment of seeds and treating seed with fungicides is often effective. The use of sugarcane varieties resistant to red rot is also recommended. The possibilities of an epidemic is very much minimized with the practice of long crop rotations (2 to 3 years) where planting is done in plots. One of the best ways to reduce the incidence of the disease is to raise healthy stock for planting in plots especially fertilized, cultivated, and kept disease-free by constant care.

 

Early blight of potato

Organism–Alternaria solani,

 

Early blight is a common foliage disease of potato and tomato. It is of common occurrence both in cold as well as in warm regions in India and abroad where, potatoes and tomatoes are grown. The pathogen causes injury to the leaves as a leaf spot disease and lead to premature defoliation. Finally it causes tuber rot of potato and fruit rot of tomato.

 Symptoms: 

• The disease appears on the leaflets, 3-4 weeks after the crop is sown as small, isolated, scattered pale brown to dark spots, oval or angular in shape mostly up to 3 or 4 mm in diameter.
• The chlorotic zone increases with the increase in size of the spot. As a rule the oldest (lowest) leaves are affected first and the disease progresses upwards.
• The necrotic tissue of the spot often shows a series of concentric ridges, a symptom characteristic of this disease
• The number of spots on the leaflets may be a few but if the conditions are favourable the spots increase in number and size involving the entire leaf surface.
• In the advanced stage when the number of spots is numerous, the leaf shows signs of old age and droops.
• The spots become hard in dry weather and the leaflets curl. Under humid conditions, the diseased areas coalesce and big rotting patches appear on the leaf surface.
• In severe cases of infection the leaves dry up, shrivel and drop off.
• Falling of leaves starts with the older (lower) ones until a few remain at the top. 
• Stems and petioles may also develop brown to dark lesions which may finally lead to collapse of the entire over-ground portion of the plant.
• Further, there isinfection and rotting of potato tubers. The surface lesions on the potato tubers are a little darker than the healthy skin and slightly sunken. They are irregular or circular in shape. The older lesions develop fissures.
• The injury to the foliage may lead to premature defoliation reduces photosynthetic activity of the plant. Consequently the tubers formed remain small and a few in number. This reduces crop yield. The disease alos result in rotting of tubers.

 

Symptoms on Leaves: The disease can be seen as small, scattered, pale brown spots on the leaflets. These spots are covered with growth of the fungus.

Necrotic Area: In the necrotic area of the spots concentric ridges develop to produce a characteristics board effect.

Target Board: ‘Target Board’ is the most characteristics symptom of early blight of potato.

Vein Chlorosis: Later, vein chlorosis may also occur.

Curling of Leaves: In dry weather the spots turn hard and the leaves curling can be seen.

Rotting of Leaves: In humid weather big totting patches appear on leaf surface.

Severe Attack: Leaves shrivel and fall off. Stems show brown to black necrotic lesions on the skin. Rotten plant may collapse.

 

Transmission

 

Early Blight of Potato is very common disease of potato in India. The occurrence of the disease can be seen few weeks after sowing the tubers. But, the damage to the crop is more during the tuber formation. The disease can be seen both in the cool and warm areas. Farmers of hills and plains, both face this serious disease.

·       The fungus remains viable in infected leaves for many years.

·       Conidia and mycelium survive in the soil on plant debris to cause primary infection to next crop.

·       Contaminated tubers also act as a source of primary infection. 

• Climatic or soil conditions which are unfavourable to the host and reduce its physiological vigour tend to promote the disease.
• The disease spreads and becomes serious when the season begins with abundant moisture followed by high temperatures because these conditions are unfavourable to the host.
• Weaker plants are more susceptible to disease than the healthier ones.
• High soil fertility tends to reduce the severity of disease.
• Periods of continued drought also check its spread.

 Management

 

·       As in the case of other soil borne diseases, crop rotation and field sanitation are the two rational measures which provide effective check against primary infection from spores in the infected plant debris.

·       Infected dry leaves and dead plants should be burned immediately after harvest.

·       Application of regular sprays (with Bordeaux mixture or other suitable fungicides) throughout the period of plant growth effectively control the early blight.

 

 

Bordeaux mixture is a fungicide made of copper sulphate and calcium hydroxide (lime). Bordeaux mixture—a combination of copper sulfate, lime, and water—is an effective fungicide and bactericide used for decades to control diseases of fruit and nut trees, vine fruits, and ornamental plants.

It is widely used to control potato blight, peach leaf curl and apple scab.

Bordeaux mixture has copper ions (Cu²⁺) which inhibit fungal enzymes and prevent germination. It is used preventively, before the fungal disease occurs. It is sprayed on the plants usually once a year.

Because the copper ions build up in the soil, continuous use will cause heavy metal pollution. Copper also bioaccumulates in organisms. So its use is restricted in some countries

 

Fungal Diseases - Fusarium Wilt, Wilt of cotton

 

Diseases are a major limiting factor for crop production. Diseases can be caused by infectious microorganisms that include fungi, bacteria, viruses and nematodes. These diseases are contagious and can spread from plant to plant in a field, often very rapidly when environmental conditions are favorable.  

1) Fusarium Wilt

Organism - Fusarium oxysporum f.sp. lycopersici 

Wilt diseases are caused by pathogens that invade the vascular system (xylem tissue) and disrupt water flow through the plant. Fusarium wilt is the major wilt disease of tomato.  This is a warm-weather disease caused by the fungus Fusarium oxysporum.

Symptom

• The first symptom of the disease is drooping and wilting of lower leaves with a loss of green colour (chlorosis)
• Affected leaflets wilt and die. The younger leaves may die in succession and symptoms continue in subsequent leaves.
• Often leaves on only one side of the stem turn golden yellow at first.
• The disease progresses up the stem. At later stage, browning of vascular system occurs.
•  The stem of wilted plants shows no soft decay, but when cut lengthwise, the lower stem will have a dark brown discoloration of the water-conducting vessels.
• Soon the petiole and the leaves droop and wilt until all of the foliage is killed and the plant becomes stunted and dies.

Transmission

• The fungus is soil-borne and passes upward from the roots into the water-conducting system of the stem. Blocking of the water-conducting vessels is the main reason for wilting.
• The fungus survives and persists indefinitely in field soil.
• Invasion occurs through wounds in roots growing through infested soil.
• Long-distance spread is through seed and transplants.
• Soils become infested by planting infected transplants and from movement of infested soil by wind and water erosion or on farm implements.

Prevention & Treatment:

• The affected plants should be removed and destroyed.
• For control, grow plants in pathogen-free soil
• Use disease-free transplants
• Grow only cultivars with resistance to Fusarium wilt. Growing tomato varieties resistant to Fusarium wilt is the most effective means of control.
• Crop rotation with a non-host crop such as cereals is also an effective control method
• Spot drench with Carbendazim (0.1%)

 

2) Wilt of cotton

Organism - Fusarium oxysporum f.sp. vasinfectum & Verticillium dahlia

 

Vascular wilts, such as Fusarium wilt and Verticillium wilt, are capable of significantly reducing cotton yields, and negatively impacting fiber quality. Properly diagnosing these diseases is critical in developing a management system. While subtle differences can be observed in the field, laboratory examinations are often required to differentiate the two diseases. Variety selection is the most effective factor in reducing wilt associated losses.

Fusarium wilt

Causal agent: Fusarium oxysporum f. sp. vasinfectum (Fov)

Disease development depends on host susceptibility and pathogen - infection by Fov is more if root-knot nematode (Meloidogyne incognita)  infection is present.  Disease occurs in warmer temperatures and sandy soils.

Fusarium can cause severe symptoms in susceptible cotton varieties. These include a general wilt and yellowing and necrosis of lower leaf margins. The vascular system of infected plants is discolored brown in affected portions of the tissue. This is most apparent in the lower stem and upper taproot. The discoloration starts in the taproot, spreads into the stem, and is generally continuous in contrast to the speckling nature of the discoloration in plants affected by Verticillium wilt.

Symptoms

occur throughout the growing season

• Initial symptoms consist of chlorosis and wilting on the margin of the leaf

• Infected seedlings often die, and may be confused with seedling disease. In seedlings and young plants, cotyledons and leaves wilt, may turn necrotic, and even fall off the plant, resulting in bare stems.

• Diseased plants exhibit a continuous discoloration of the vascular tissue

· Seedlings of susceptible varieties often die and in mildly affected plants, lower leaves develop symptoms but plants survive, but with reduced vigor and noticeable stunting.

• Fusarium wilt occur more if root-knot nematode infection is present. Certain strains of the causal fungus only cause symptoms when plants are also infected with the root knot nematode. In those cases, galls are usually prevalent on lateral roots.

 

 

Transmission

·       The fungus sustains itself on the outer surface of roots of many crops and weeds and survives indefinitely in soil.

·       The pathogen is seed-borne in cotton, causing long-distance spread,

·      Is also spread whenever infested soil is transported on boots, farm equipment, in flood irrigation, etc.

 

Verticillium wilt

Organism: Verticillium dahliae

Disease development depends on host: variety, developmental stage, and plant density, pathogen: defoliating vs. non- defoliating type, aggressiveness, and inoculum density. Occurs in  cool, wet environments

Symptoms

• Initial root infections occur early; symptoms are more evident post-bloom

• Infected plants may be wilted or stunted

• Chlorosis or necrosis of leaf margins and interveinal areas may be

• Light to dark brown discoloration (speckled) is prominent in the vascular system

• Severe defoliation can result, leading to reductions in yield and fiber quality

        

Management of cotton wilt

• Proper identification of pathogen is required. Sample infected plants for presence of  Fov / Verticillium

• Use pathogen-free seed produced in disease-free fields at all times

• Assay soil for root-knot nematodes- Nematode risk level will influence management options-  Use of nematicides will indirectly lead to less Fusarium wilt damage. Control of the root knot nematode is important to managing Fusarium wilt caused by most genotypes of the causal fungus. Nematicides, root knot nematode-resistant varieties or both are often necessary in fields infested with the nematode

• Rotation to any crop other than cotton prevents an increase in the soil population of Fusarium but may not significantly reduce the number of spores in the soil. The fungus will sustain itself on the roots of most plants, including weeds (without causing any symptoms), and cannot be eliminated by crop rotation alone.

• Remove infected plants. Use disease resistant varieties/cultivars for several seasons to reduce wilt severity in subsequent years

• Any field operation that moves soil from one location to another can spread spores of the fungus and introduce it to other fields. Washing soil from equipment with pressurized water will help limit the spread of Fusarium

• Other containment options include restricting traffic in affected patches, destroying affected plants and surrounding non-symptomatic plants, and stopping irrigation of affected patches in order to prevent movement of infested soil.