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Pythium deliense causing severe damping-off of cucumber seedlings and its biological control by soil bacteria
Microbiol. Res. (1996) 151,309-312
Microbiological Research © Gustav Fischer Verlag Jena
Pythium deliense causing severe damping-off of cucumber seedlings and its biological control by soil bacteria Bernard Paul, Delphine Bazireau, Gerard Gambade Laboratoire de Mycologie et de biodiversite, U.F.R. Sciences de la vie, Universite de Bourgogne, B.P. 138,21004 Dijon, France Accepted: February 11, 1996
Abstract Pythium deliense Meurs was isolated from cucumber plant debris and was found to be highly pathogenic to cucumber seedlings, causing severe damping-off disease. The fungus however was completely inhibited by a soil bacterium SU-48.1. In vitro and in vivo studies reveal that the disease can be controlled by using this bacterium. The details of the parasitic fungus, the antagonism between the fungus and the bacteria, and the biological control of the disease, are described in this article. Key words: Pythium - bacteria - antagonisms - damping-off - biological control
Introduction The members of the genus Pythium are well known plant pathogens throughout the world. Apart from seed, root, and fruit rots; feeder root necrosis, and decline diseases, these fungi are the causal organisms of the damping-off disease which results in the sudden toppling of young seedlings in nursery beds, greenhouse flats, and row crops (Hendrix and Campbell 1973). Pythium deliense was first described by Meurs (1934) isolated in Sumatra from a tobacco plant suffering from stem-burn disease. Since then it has been isolated from warmer countries and is knownto be pathogenic to tobacco, cucurbits, maize, tomato, and ginger (Plaats-Niterink 1981). It has not been reported from colder countries. This is the first report of its presence in the south western France, and its biological control with the help of soil bacteria. Corresponding author: B. Paul
The fungus is easily recognizable due to its filamentous inflated sporangia, its very special oogonial stalks bending towards the antheridia, and aplerotic oospores. The control of damping-off disease till now, has been mainly chemical. Too much of fungicides have been employed in the past, and the ill effects of the chemical treatment to the environment is becoming increasingly apparent. The appearance of resistant varieties of plant pathogens is yet another consequence of the massive utilisation of chemicals. During recent years efforts are being made towards the biological control of soil borne diseases. A brief account of the biological control of pythiaceous fungi has already been discussed (Paul 1995}. In this study repeated experiments have shown that damping-off disease of cucumber caused by Pythium deliense can effectively be controlled by using the soil bacterium SU-48.1.
Materials and methods Bacterial strain SU -48.1 was isolated from a soil sample collected in Alma-Ata (Kazakastan). This was grown on nutritive agar slants and in Trypcasesoya broth, and incubated at 28°C. Pythium deliense (F-331) was isolated from plant debris collected together with soil samples in Perpignan in the south-western France. It was cultured on boiled hemp-seed halves in sterile distilled water, and on solid media like potato carrot agar (PCA) or corn meal agar (CMA). These were incubated at different temperatures for sporulation. To observe the antagonism between the fungal and the bacterial isolates, Microbiol. Res. 151 (1996) 3
309
6 ••••
4-
......
~
Fig. 1-21. Pythium
@
21
both were inoculated on the same PCA plate on opposite ends. The plate was then incubated at 25 °C. The inoculation experiments with cucumber seeds were done as described earlier (Paul 1995) using three sets of plastic pots, one acting as control, the other for fungul inoculation, and the third one for fungal and bacterial inoculation. The experiment was repeated several times. The fungus (F-331) was identified with the help of keys provided by Middleton (1943), Waterhouse (1967), Plaats-Niterink (1981), and Dick (1990). Both, the fungal and the bacterial isolates are maintained at the Laboratoire de Mycologie, University of Bourgogne, Dijon, France. 310
Microbiol. Res. 151 (1996) 3
deliense;
Fig. 1-3: filamentous inflated sporangia. Fig. 4-8: oogonial stalks bending towards antheridia. Fig. 9 - 11 : antheridial stalks bending towards oogonia. Fig. 12 -13: oogonia and intercalary antheridia. Fig. 14-15: developing oospores. Fig. 16-20: aplerotic oospores. Fig. 21: plerotic oospore. Figures 1- 21 bar = 25 lim
Observations and results The/ungus (F-331) Main hyphae are 7 - 8 ~m diam., Sporangia composed of filamentous inflated elements with inflated lateral branches. Zoospores formed at 25°C. The encysed ones measure upto 13 ~m in diameter. Oogonia smooth walled, terminal, 14- 30 ~m (av. 24.4) diam., oogonial stalks often bending towards the antheridia. Antheridia mostly monoclinous, usually one per oogonium, terminal or intercalary, antheridial cells large making apical contact with the female gametangial wall. The antheridial stalks are usually
22
Fig. 22-23. Cucumber seedlings. a: helathy seedling; b: cucumber seeds + Pythium deliense damping-off; c: cucumber seeds + Pythium deliense + bacteria SU-48.1 - healthy seedling.
pre-emergence
Fig. 24. Bacterium SU-48.1 and its spores. Fig. 25-27. Pythium deliense; Fig. 25: filamentous inflated sporangia. Fig. 26: antheridia and oogonia. Fig. 27: developping oospores. Figures 22 - 27 bar = 30 ~m
Microbiol. Res. 151 (1996) 3
311
straight but at times these tend to bend towards oogonia too. Oospores globose, aplerotic, very rarely plerotic in the smaller oogonia, smooth walled, one per oogonium, measuring 13-25 ~m (av.20.6) diam., provided with a 2 - 3 ~m thick wall. The fungus grows luxuriantly on boiled hemp seed halves in water, and on solid media. Colonies on PCA and CMA are partially submerged and partially aerial, giving a cottony appearance. On CMA the cottony aspect is more dense. The daily growth rate of the fungus on PCA at 25°C is 30 mm, while on CMA it is 35 mm at the same temperature. The Bacteria (SU-4B.l)
The antagonist bacteria (SU-23) were isolated from a soil sample taken in Alma-Ata (Kazakastan). The bacterial cells are rod shaped measuring 3 - 6 ~m in length and 1-1.25 ~m in width; and are Gram negative mobile, producing an ellipsoidal central spore measuring 2.5 ~m in length and 1.25 -1.5 ~m in width. Antagonism Antagonism between the fungus (F-331) and the
bacterial strain (SU-48.1) was observed on the PCA plates inoculated with both these organisms. The fungus grows rapidly to a certain extent and then suddenly stops, producing a clear zone of inhibition around the bacterial inoculum. Inoculation experiments gave the following results: Pot 1 (without fungus): Normal growth of the cu-
cumber seedlings. Pot 2 (With fungus): Pre-emergence damping-off of the cucumber seedling. Pot 3 (fungus + bacteria): Normal growth of the cucumber seedlings. Pythium deliense was recovered from the damped-off seed material, when this was transferred to PCA or CMA plates. This clearly indicates that the fungus Pythium deliense (F-331) is a parasite of cucumber seedlings bringing about pre-emergence damping-off symptoms within 4 - 5 days (pot 2), and that the bacterium SU-48.1 is its antagonist as it does not allow the fungus to express itself as a pathogen (pot 3).
312
Microbiol. Res. 151 (1996) 3
Conclusion Pythium deliense is reported for the first time in
France. Morphological features of the fungus agree well with those described for Pythium deliense in the literature. Some of the minor differences are: the occassional bending of antheridial stalks towards oogonium together with the normal ones (oogonial stalks bending towards the antheridia), variation in the oogonial and oospores sizes which is more than indicated in Meur's diagnosis: The oogonia of F-331 vary between 14-30 ~m (av. 24.4) diameter, instead of 15 - 23.1 (av. 18.2) ~m; and the oospores ranging between 13 - 25 ~m (av. 20.6) instead of 12.5-17.5 (av.14.8). In fact Middleton (1943) studied the type culture and found that the oogonial and oosporal dimensions were greater than in Meur's diagnosis (Oogonia avo 18.9 ~m, oospore avo 15.3 ~m). In this study the isolate F-331 has also been found to be highly pathogenic to cucumber seedlings bringing about a pre-emergence type of damping-off symptoms. The study also reveals that flooding the fungus with bacterial strain SU-48.1, inhibits the fungus and results in the healthy growth of the cucumber seedling. References Dick, M. W. (1990): Keys to Pythium. University of Reading Press, 64. Hendrix, F. F., Campbell, W. (1973): Pythiums as plant pathogens. Annual Review of Phytopathology 11, 77-98. Meurs, A. (1934): Parasitic stemburn of Deli tobacco. Phytopath. Z. 7, 169 -185. Middleton, J. T. (1943): The taxonomy, host range, and geographical distribution of the genus Pythium. Mem. Torrey Bot. Club 20, 1-171. Plaats-Niterink, A. J. van der (1981): Monograph of the genus Pythium. Studies in Mycology Baarn 21, 1- 242. Paul, B., Romond, C., Bhatnagar, T. (1995): Biological control of Pythium mamilla tum causing damping-ofT of cucumber seedlings by a soil bacterium, Bacillus mycoides Microbiol. Res. 150, 71- 75. Waterhouse, G. M. (1967): Key to Pythium Pringsheim, Mycological P, CMI 109, 1-15.
Microbiological Research © Gustav Fischer Verlag Jena
Pythium deliense causing severe damping-off of cucumber seedlings and its biological control by soil bacteria Bernard Paul, Delphine Bazireau, Gerard Gambade Laboratoire de Mycologie et de biodiversite, U.F.R. Sciences de la vie, Universite de Bourgogne, B.P. 138,21004 Dijon, France Accepted: February 11, 1996
Abstract Pythium deliense Meurs was isolated from cucumber plant debris and was found to be highly pathogenic to cucumber seedlings, causing severe damping-off disease. The fungus however was completely inhibited by a soil bacterium SU-48.1. In vitro and in vivo studies reveal that the disease can be controlled by using this bacterium. The details of the parasitic fungus, the antagonism between the fungus and the bacteria, and the biological control of the disease, are described in this article. Key words: Pythium - bacteria - antagonisms - damping-off - biological control
Introduction The members of the genus Pythium are well known plant pathogens throughout the world. Apart from seed, root, and fruit rots; feeder root necrosis, and decline diseases, these fungi are the causal organisms of the damping-off disease which results in the sudden toppling of young seedlings in nursery beds, greenhouse flats, and row crops (Hendrix and Campbell 1973). Pythium deliense was first described by Meurs (1934) isolated in Sumatra from a tobacco plant suffering from stem-burn disease. Since then it has been isolated from warmer countries and is knownto be pathogenic to tobacco, cucurbits, maize, tomato, and ginger (Plaats-Niterink 1981). It has not been reported from colder countries. This is the first report of its presence in the south western France, and its biological control with the help of soil bacteria. Corresponding author: B. Paul
The fungus is easily recognizable due to its filamentous inflated sporangia, its very special oogonial stalks bending towards the antheridia, and aplerotic oospores. The control of damping-off disease till now, has been mainly chemical. Too much of fungicides have been employed in the past, and the ill effects of the chemical treatment to the environment is becoming increasingly apparent. The appearance of resistant varieties of plant pathogens is yet another consequence of the massive utilisation of chemicals. During recent years efforts are being made towards the biological control of soil borne diseases. A brief account of the biological control of pythiaceous fungi has already been discussed (Paul 1995}. In this study repeated experiments have shown that damping-off disease of cucumber caused by Pythium deliense can effectively be controlled by using the soil bacterium SU-48.1.
Materials and methods Bacterial strain SU -48.1 was isolated from a soil sample collected in Alma-Ata (Kazakastan). This was grown on nutritive agar slants and in Trypcasesoya broth, and incubated at 28°C. Pythium deliense (F-331) was isolated from plant debris collected together with soil samples in Perpignan in the south-western France. It was cultured on boiled hemp-seed halves in sterile distilled water, and on solid media like potato carrot agar (PCA) or corn meal agar (CMA). These were incubated at different temperatures for sporulation. To observe the antagonism between the fungal and the bacterial isolates, Microbiol. Res. 151 (1996) 3
309
6 ••••
4-
......
~
Fig. 1-21. Pythium
@
21
both were inoculated on the same PCA plate on opposite ends. The plate was then incubated at 25 °C. The inoculation experiments with cucumber seeds were done as described earlier (Paul 1995) using three sets of plastic pots, one acting as control, the other for fungul inoculation, and the third one for fungal and bacterial inoculation. The experiment was repeated several times. The fungus (F-331) was identified with the help of keys provided by Middleton (1943), Waterhouse (1967), Plaats-Niterink (1981), and Dick (1990). Both, the fungal and the bacterial isolates are maintained at the Laboratoire de Mycologie, University of Bourgogne, Dijon, France. 310
Microbiol. Res. 151 (1996) 3
deliense;
Fig. 1-3: filamentous inflated sporangia. Fig. 4-8: oogonial stalks bending towards antheridia. Fig. 9 - 11 : antheridial stalks bending towards oogonia. Fig. 12 -13: oogonia and intercalary antheridia. Fig. 14-15: developing oospores. Fig. 16-20: aplerotic oospores. Fig. 21: plerotic oospore. Figures 1- 21 bar = 25 lim
Observations and results The/ungus (F-331) Main hyphae are 7 - 8 ~m diam., Sporangia composed of filamentous inflated elements with inflated lateral branches. Zoospores formed at 25°C. The encysed ones measure upto 13 ~m in diameter. Oogonia smooth walled, terminal, 14- 30 ~m (av. 24.4) diam., oogonial stalks often bending towards the antheridia. Antheridia mostly monoclinous, usually one per oogonium, terminal or intercalary, antheridial cells large making apical contact with the female gametangial wall. The antheridial stalks are usually
22
Fig. 22-23. Cucumber seedlings. a: helathy seedling; b: cucumber seeds + Pythium deliense damping-off; c: cucumber seeds + Pythium deliense + bacteria SU-48.1 - healthy seedling.
pre-emergence
Fig. 24. Bacterium SU-48.1 and its spores. Fig. 25-27. Pythium deliense; Fig. 25: filamentous inflated sporangia. Fig. 26: antheridia and oogonia. Fig. 27: developping oospores. Figures 22 - 27 bar = 30 ~m
Microbiol. Res. 151 (1996) 3
311
straight but at times these tend to bend towards oogonia too. Oospores globose, aplerotic, very rarely plerotic in the smaller oogonia, smooth walled, one per oogonium, measuring 13-25 ~m (av.20.6) diam., provided with a 2 - 3 ~m thick wall. The fungus grows luxuriantly on boiled hemp seed halves in water, and on solid media. Colonies on PCA and CMA are partially submerged and partially aerial, giving a cottony appearance. On CMA the cottony aspect is more dense. The daily growth rate of the fungus on PCA at 25°C is 30 mm, while on CMA it is 35 mm at the same temperature. The Bacteria (SU-4B.l)
The antagonist bacteria (SU-23) were isolated from a soil sample taken in Alma-Ata (Kazakastan). The bacterial cells are rod shaped measuring 3 - 6 ~m in length and 1-1.25 ~m in width; and are Gram negative mobile, producing an ellipsoidal central spore measuring 2.5 ~m in length and 1.25 -1.5 ~m in width. Antagonism Antagonism between the fungus (F-331) and the
bacterial strain (SU-48.1) was observed on the PCA plates inoculated with both these organisms. The fungus grows rapidly to a certain extent and then suddenly stops, producing a clear zone of inhibition around the bacterial inoculum. Inoculation experiments gave the following results: Pot 1 (without fungus): Normal growth of the cu-
cumber seedlings. Pot 2 (With fungus): Pre-emergence damping-off of the cucumber seedling. Pot 3 (fungus + bacteria): Normal growth of the cucumber seedlings. Pythium deliense was recovered from the damped-off seed material, when this was transferred to PCA or CMA plates. This clearly indicates that the fungus Pythium deliense (F-331) is a parasite of cucumber seedlings bringing about pre-emergence damping-off symptoms within 4 - 5 days (pot 2), and that the bacterium SU-48.1 is its antagonist as it does not allow the fungus to express itself as a pathogen (pot 3).
312
Microbiol. Res. 151 (1996) 3
Conclusion Pythium deliense is reported for the first time in
France. Morphological features of the fungus agree well with those described for Pythium deliense in the literature. Some of the minor differences are: the occassional bending of antheridial stalks towards oogonium together with the normal ones (oogonial stalks bending towards the antheridia), variation in the oogonial and oospores sizes which is more than indicated in Meur's diagnosis: The oogonia of F-331 vary between 14-30 ~m (av. 24.4) diameter, instead of 15 - 23.1 (av. 18.2) ~m; and the oospores ranging between 13 - 25 ~m (av. 20.6) instead of 12.5-17.5 (av.14.8). In fact Middleton (1943) studied the type culture and found that the oogonial and oosporal dimensions were greater than in Meur's diagnosis (Oogonia avo 18.9 ~m, oospore avo 15.3 ~m). In this study the isolate F-331 has also been found to be highly pathogenic to cucumber seedlings bringing about a pre-emergence type of damping-off symptoms. The study also reveals that flooding the fungus with bacterial strain SU-48.1, inhibits the fungus and results in the healthy growth of the cucumber seedling. References Dick, M. W. (1990): Keys to Pythium. University of Reading Press, 64. Hendrix, F. F., Campbell, W. (1973): Pythiums as plant pathogens. Annual Review of Phytopathology 11, 77-98. Meurs, A. (1934): Parasitic stemburn of Deli tobacco. Phytopath. Z. 7, 169 -185. Middleton, J. T. (1943): The taxonomy, host range, and geographical distribution of the genus Pythium. Mem. Torrey Bot. Club 20, 1-171. Plaats-Niterink, A. J. van der (1981): Monograph of the genus Pythium. Studies in Mycology Baarn 21, 1- 242. Paul, B., Romond, C., Bhatnagar, T. (1995): Biological control of Pythium mamilla tum causing damping-ofT of cucumber seedlings by a soil bacterium, Bacillus mycoides Microbiol. Res. 150, 71- 75. Waterhouse, G. M. (1967): Key to Pythium Pringsheim, Mycological P, CMI 109, 1-15.