Occurrence of Pseudomonas aeruginosa (Schroeter) Migula as a pathogenic bacterium of the desert locust, Schistocerca gregaria (Forskål)

Occurrence of Pseudomonas aeruginosa (Schroeter) Migula as a pathogenic bacterium of the desert locust, Schistocerca gregaria (Forskål)

JOURNAL OF INVERTEBRATE PATHOLOGY Occurrence Migula of (1965) Pseudomonas aeruginosa as a Pathogenic Locust, SHAHID Pesticide 7, 189-191 Re...

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Occurrence Migula





as a Pathogenic Locust,

SHAHID Pesticide

7, 189-191



Schistocercu H.




gregaria I.


(Sehroeter) the

(ForskAl) AND


Section, Central Laboratories, Pakistan Council altd Industrial Reseavck, Karachi, Pakistan Accepted




of Scientijirc

20, 1964

aeruginosa (Schroeter) Migula was isolated from the desert locust, gregaria (Forskal), collected in the Nabisar area of West Pakistan in 1962. The bacterium designated as “strain CL” was identified by morphological and cultural characters. The median lethal dose for locusts was about 33 bacteria injected into the hemocoel, or about 21,COO bacteria ingested with the food. The bacterium was not infectious for rats by oral or subcutaneous routes. Pseudomonas Sckistocerca

pigment, were observed. The identification of Ashrafi and Ghayasuddin (1962) reported the bacterium was based on its staining propthe presence of Serratia maycescens Bizio, erties, cultural characteristics, and biochemical reactions. “Bergey’s Manual of DetermiBacillus subtilis Cohn, Bacillus ceyeusFranknative Bacteriology” (Breed et al., 1957) was land and Frankland, and Staphylococcus pyogenesauleus Rosenbachin the desert locust, followed for confirmation of results. The bacSchistocerca gregaria (Forskil). Later three terium was a gram-negative, motile coccodifferent fungi, Aspergillus ustus (Bain) Thorn bacillus. CoIonies of the bacterium, on nutrient-agar plates, were slightly raised and and Church, Helminthosporium hawaiiense Bugnicourt, and Aspergillus fEavus Link, and easily emulsifiable in water. They varied from 1 mm to 7 mm in diameter. The bacterium a bacterium identified as Pseudomonas aerugiproduced a granular surface pellicle and a nosa (Schroeter) Migula were isolated from heavy deposit in broth. It fermented glucose fifth nymphal instars of the desert locust. and xylose without gas production and did ISOL.~TI~N AND IDENTIFX~TION not ferment lactose, maltose, sucrose, arabinose, rhamnose, mannose,mannitol, inositol, Sick fifth nymphal instars of Schistocerca gregaria collected from Nabisar area of West and inulin. It liquefied gelatin, produced acid Pakistan were brought to the laboratories. and clot in litmus milk, utilized citrate, and These were sterilized on the outer surface and did not produce H&S. Acetylmethylcarbinol each locust was triturated in 10 ml of sterile was not formed. Indole was not produced. distilled water. Serial dilutions in the range of The methyl red test was negative; methylene 10-l to 10-G were made; 0.1 ml of suspen- blue was not reduced. sion from each dilution was plated out on The bacterium was inoculated in paraffmnutrient agar. The plates were maintained at covered broth tubes and incubated for a week 25°C; after 24 hours almost pure cultures of at 37°C. It failed to grow in anaerobic cona bacterium, which produced diffused green ditions, showing that it was strictly aerobic. 189




The bacterium grew between pH 5 to pH 11 with an optimum pH 7.6. Irrespective of the initial pH, the organism changed the pH of all broth tubes to 7.4-7.6. Observations based on the above characteristics gave confirmation that the bacterium was Pseudomonas aeruginosa (Schroeter) Migula, and it was designatedas “strain CL.”



organisms/ml. From this diluted suspension further dilutions in the range of 0.1: 0.25, 0.5, 0.75, and 1 percent were prepared: 0.02 ml of each of the above dilutions was inoculated into batches of 10 locusts each. Controls were run in which the same quantity of sterile distilled water and broth were inoculated. Percent mortality was noted after 24 hours. Log doses against probit mortality were PATHOGENICITY plotted to calculate the LDno value, which Before using healthy locusts for patho- was found to range between 27 to 33 bacteria,/ genicity experiments, their hemolymph was locust. examined and those found free from Pseudomonas aeruginosa infection were used in the Food Injectiorz experiments. Then Pseudomonas aeruginosa A 24-hour broth culture of Pseudomonas “strain CL” was intrahemocoelically inocu- aeruginosa was diluted in sterile distilled water lated. The locusts died after 24 hours with to contain bacteria ranging from 10.000 to the characteristic symptoms of the disease, 100,000 per ml; 0.5 ml of each dilution was i.e., the body turned reddish in color. The used to infect 2 g of cabbage leaves. Locusts appendages and other parts of the body were starved for 24 hours, then released on showedgreat degeneration and disintegration. the contaminated food. Percent mortality was From the dead locusts Pseudomonas aevuginoted after 48 hours. Log dosesagainst probit nosawas reisolated in almost pure culture and mortality were plotted and the LDao value was used again to causethe sameinfection in was calculated to lie between 18,000 to 24,000 healthy locusts. This proved that Pseudobacteria/locust. monas aeruginosa was the causative agent of Direct Spraying the diseasein the desert locust. Jars of 2-liter capacity were taken. In each After establishing the pathogenicity of the bacterium against the desert locust, experi- jar five locusts were kept. A 24-hour broth aeruginosa “strain ments were run to determine the lethal doses culture of Pseudomonas and the time required by the bacteria to CL” was diluted in sterile distilled water to reach various parts of the gut after hemocoelic contain approximately 528,000 bacteria/ml. infection. The locusts were infected by three From this suspension,0.5 ml was sprayed on methods: first: by intrahemocoelic inocula- locusts in each jar by a DeVilbiss hand atomtion ; second, through food infection; and izer. After 10 minutes the locusts were transthird, by direct bacterial spraying on locusts. ferred into 10 holding cages.The experiments were divided in two batches of locusts. One Intrahemocoelic Inoculation batch was kept at 38 2 2°C and 35 t 5% Locusts were inoculated between the second R.H., and showed 100 percent mortality after and third tergum of the abdomen. The inocu- 24 hours while the other batch, kept at lations were carried out with an Agla microm- 25 ? 2°C and 85 i 5% R.H., showed 100 eter syringe outfit which delivers a volume of percent mortality after 48 hours. 0.01 ml in one complete revolution of the micrometer head (Trevan, 1925). A 24-hour Bacteria2 Occurrence in the Gut after Hemolymph injection broth culture of Pseudomonas aeruginosa Pseudomonas aeruginosa “strain CL” was “strain CL” was diluted in sterile distilled water to contain from 750,000 to l,OOO,OOO inoculated into the hemocoel of adult male



healthy locusts and experiments were run to determine the time taken by the bacteria to reach the various parts of the gut from hemocoel. A heavy dose of 0.02 ml suspension containing 3,000,OOO bacteria/ml was inoculated into the hemocoel to cause a fatal infection within 24 hours. Inoculated batches of locusts were dissected out after every hour and the contents of the foregut, midgut, and hindgut of each locust were plated out and incubated at 37°C for 24 hours. The bacteria were found repeatedly in the foregut after 4 hours, in the midgut after 6 hours, and in the hindgut after 5 hours of hemolymph infection. Infectivity

for Rats

Four batches of five rats each were fed in food with 1 ml, 2 ml, 4 ml, and 6 ml of a 24hour broth culture of Pseudomonas aeruginosa diluted to approximately 40,000 bacteria/ml. The rats developed no infection over a period of 3 months. Five batches of five rats each were inoculated subcutaneously with 0.1 ml, 0.2 ml, 0.5 ml, 0.75 ml, and 1 ml/kg body weight of a 24-hour-old broth culture diluted to 440,000 bacteria/ml. None of the rats developed infection. DISCUSSION

Pseudomonas aeruginosa “strain CL” isolated from wild desert locusts, fermented glucoseand xylose but did not ferment mannose,




which was fermented by the strain of this bacterium isolated from grasshoppers by Bucher and Stephens (1957). The optimum pH range of the medium for culturing the bacteria was found to be 7.4 to 7.6 which was different from that of the bacterium reported from grasshoppers. It was found that bacteria took less time to reach the foregut and hindgut from the hemolymph than to reach the midgut. Hence it may be suggested that the bacteria have directly passed from the hemolymph to the foregut and hindgut rather than entering into foregut and passing to midgut and then to hindgut. .‘,CKNOWLEDGMESTS

The authors are highly indebted zaman Siddique, F.R.S., Chairman, rachi (Pakistan) for encouragement during the progress of the work paper.

to Dr. SalimmuzP.C.S.I.R., Kaand suggestions described in this


ASHRAFI, S. H., AND GHAYASUDDIN, M. 1962. Pathogenic bacteria of the desert locust, Schistocerca gregaria. Pakistan J. Sci. Ind. Rex., 5, 42-44. BREED, R. S., MURRAY, E. G. D., AND SMITH, N. R. 1957. “Bergey’s Manual of Determinative Bacteriology,” 7th ed., 1094 pp. Williams & Wilkins, Baltimore, Maryland. BUCKER, G. E., AND STEPHENS, J. M. 1957. A disease of grasshoppers caused by the bacterium Pseudomonas aeruginosa (Schroeter) Migula. Can. J. Microbial., 3, 611-625. TREVAN, J. W. 1925. The micrometer syringe. Bio&em. J., 19, 1111-1114.