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Fungus flora of Coffea arabica in Nyasaland
[ 281 ] Trans. Brit. mycol. Soc. 47 (2),281-284 (1964) Printed in Great Britain
FUNGUS FLORA OF COFFEA ARABICA IN NYASALAND By M. A. SIDDIQI Department of Agriculture, Nyasaland The fungi isolated from all parts of the plant and surroundings are listed. From six coffee sources, 81 species of fungi were isolated. The distribution of the saprophytic and pathogenic isolates is discussed.
A large amount of literature on coffee diseases exists, the most recent contribution being by Wellman (1961) in which a full survey of coffee pathology is made. While much is known about coffee diseases and their distribution, the microflora of coffee has received less attention. During a study of the bark diseases of CojJea arabica in Nyasaland during 1959 to 1962 (Siddiqi & Corbett, 1963), a record was kept of the fungi isolated from the soil, roots, and aerial parts and from a spore trap in coffee fields, and the results of this exercise are presented here. MATERIALS AND METHODS
The coffee gardens in which these observations were made are all in the coffee-growing areas of Nyasaland. Coffee soils are generally moderately acid (top soil pH 5"6-6'4) with a more acid subsoil. They are deep red sandy-clay loams usually well drained but occasionally impeded by a lateritic gravel layer at depth.
Collection of samples and isolations The numbers of samples and frequency with which they were taken from various coffee gardens were not constant because the samples were originally intended for a study of the diseases caused by a form of Fusarium stilboides Wollenw. (Siddiqi & Corbett, 1963). All isolations were made on a standard agar medium with the following composition: glucose, IO g.; KN0 3 , 1 g.; KH 2P0 4 , I g.; agar, 20 g.; rose bengal, 67 p.p.m.; tapwater, 1 1. The pH of the medium was adjusted to 6. Sometimes the standard agar medium was used without rose bengal, which was added to inhibit bacterial development (Smith & Dawson, 1944); it is very efficient in this respect and also has the advantage offavouring a more varied fungus flora than does the use of a very acid medium. The general procedure for collecting samples and making isolations (Table I) was as follows: (I) Soil. Three random samples of soil were taken to approximately 8 in. depth using a trowel. The three samples were thoroughly mixed and passed through a 2 mm. sieve. The soil was incorporated in melted agar medium (approximately 5 mg. per IO ml. medium) cooled to 50° C. and the plates poured immediately (Warcup, 1950). 18
MYC.47
Transactions British Mycological Society
282
(2) Roots. Three root samples were taken from each unhealthy plant examined. They were thoroughly washed to remove adhering soil. From these, twenty-five rootlets were selected at random and cut into small pieces. The pieces were mixed thoroughly and twenty-four pieces selected at random, surface sterilized by dipping in absolute alcohol for 30 sec., washed in sterile water and then placed on the surface of standard agar plates. (3) Bark andbranches. Pieces of infected bark and branches were surface sterilized in o· 1 % mercuric chloride solution or absolute alcohol, washed in sterile water and then placed on the surface of standard agar plates. (4) Leaves and fruit. After incubation in moist chambers these were examined directly and pieces were also plated on the standard agar medium. (5) Spore trap. A simple spore trap (Stephen, 1957) was used, placed in the middle of each coffee garden. As it was difficult to identify fungi solely from the spores on the slides by direct examination, cultures were made. Each slide was placed in a sterile Petri dish and 15 ml. standard agar medium cooled to 50° C. poured on top. Recording offungi Plates were incubated at room temperature (20-25° C.) and kept under observation for 6 weeks. Forms found difficult to identify directly by microscopic examination of the plates were isolated in pure culture. Fungi which did not sporulate are designated as unidentified species. NOTES ON THE OCCURRENCE OF THE FUNGI
From the six areas a total of eighty-one specieswas isolated and identified (Table 2). Because the isolates were originally taken for specific studies of coffee bark diseases (Siddiqi & Corbett, 1963) selective media or techniques were not used. If this had been done additional species of fungi would probably have been obtained. Table
I.
Analysis of samples, plates examined andfungi identified, August, 1959-Ju(Y, 1962 No. of samples
Bark and branches Roots Leaves Fruit Soil Spore trap
No. of plates
Saprophytic spp.
Pathogenic spp.
4
8
346
1730
100 500 300
500
78
780
54
128
128
31
4 I 2
5 9 5 4 5
The distribution of saprophytic and known pathogenic fungi is given in Table I. High numbers of saprophytic fungi occur in the soil and on spore trap slides. The few fungi isolated from roots and the aerial parts of coffee are mostly pathogenic (Table 2, P) and all are well-known coffee pathogens reported from other coffee-growing countries as well. The few saprophytes isolated from roots and aerial parts were secondary invaders.
28 3
Fungus flora of coffee. M. A. Siddiqi Table
2.
List offungi isolated from the different sources Soil
Absidia sp. Circinella simplex van Tiegh. Cunninghamella sp. Mortierella pusilla Oud. Mortierella sp. Mucor sp. M. racemosus Fresen. Rhizopus sp. Zygorrhynchus sp. Capnodium brasiliense Putten Chaetomium cochlioides Pall. Chaetomium sp. Mycosphaerella coffeae Noack Nematospora sp, Pseudogymnoascus vinaceus Raillo Thielavia terricola (Gilm. & Abb.) Emmons Westerdykella sp. Acremonium sp. Acrothecium sp. Alternaria sp. Arthrobotrys sp. Ascochyta sp Aspergillus fumigatus Fresen. A. flaous Link ex Fr. A. niger van Tiegh. Blastomyces sp. Botryotrichum sp. Botrytiscinerea Pers. ex Fr. Cephalosporium acremonium Corda Cercospora coffeicola Berk. & Cooke Cladosporium herbarum (Pers.) Link ex Fr. Cladosporium sp. Colletotrichum coffeanum Noack Curvularia sp. Cylindrocarpon sp. Diplodia sp. Fusarium solani (Mart.) Sacco F. equiseti (Corda) Sacco F. oxysporum Schlecht. ex Fries F. stilboides Wollenw. Geotrichum sp, Gliocladium roseum Bain. Helminthosporium sp. Hormiscium sp. Hormodendrum cladosporioides (Fresen.) De Vries Mycogone sp, Nigrospora sphaerica (Sacc.) Mason Oospora sp. O. egyptiaca van Beyma Papularia sp. Penicillium spp.
Bark and branches
Leaves
Fruit
+ + + + + + + + P P -
+ + +
+ +
+
+
+
+
+
+
+
P
+ + + +
P P
+ +
+
+ + + + + + +
+ +
+ + + + + + + + +
+
+
+ + + + + +
P
P P P
+
+
P
P
+ +
+ +
P
Spore trap
+ +
+ +
(5 spp.) Pestalotia sp. Phoma sp, Pullularia sp.
Roots
+ + +
+ +
+
+ +
+ + + + + + + + +
+ +
+
+
(I sp.)
+ +
(I sp.)
+
+ + +
(3 spp.)
18-2
28 4
Transactions British Mycological Society Table Soil
Sepedonium sp, Sphaeronaema sp. Spicaria violacea Abbott Thielaoiopsis sp. Ti lachlidium sp. Torula lucif uga Oud. Trichoderma viride Pers. ex Fr. T. glaucum Abbott T. koningii Oud, Trichophy ton sp. Verticillium terrestre (Link) Lindau Armillaria mellea (Vah!. ex Fr. ) Kummer Clito/iYbe sp , Hemileia vastatrix Berk, & Br. (Race II) Mycena sp. Pellicularia koleroga Cooke Rhizoctonia sp. Un identified spp.
+ + + + + + + + + +
Present .
Bark and branches
Leaves
+ +
+ +
Fruit
Spore trap
+ + +
+
(6 spp.) 58
+
Roots
+
P P
Totals
(cont).
+
P
P P P
2
+
+ + +
+ + +
9
12
10
+ +
7
(3 spp ) 36
P = pa thogenic on coffee.
The most common fungi isolate d from soil (Table 2) were Aspergillus fumigatus , A. niger, Trichoderma viride, T . koningii, Cephalosporium acremonium, Cladosporium herbarum, Cladosporium sp., Fusarium solani, F. oxy sporum, F. equiseti, Penicillium spp. and Cunninghamella sp. These, with the exception of F. oxysporum, were also common on th e spore trap slides. None of the saprophytic or pathogenic fungi was pr evalent in all the six coffee habitats studied. I wish to thank Mr D. C. M. Corbett , Department of Agriculture, Nyasaland, for helpful criticism in the preparation of this paper, the late Dr W. L. Gordon, Can ad a Dep artment of Agriculture , for confirming th e identity of Fusarium cultures, and the Director and Sta ff of th e Commonwealth Mycological Institute, K ew, for looking through th e list of fungi. This paper is published by permission of the Director of Agriculture and Fisherie s, Nyasaland. REFERENCES
SIDDIQ.I, M . A. & CORBETI, D . C. M . (1963) . Coffee bark diseases in Nyasala nd. I. Pathogeni city, description and identity of th e causal orga nism. Tr ans. B rit. mycol. Soc. 46, 9 1-IO I. S~fiTH, N. R . & DAWSON, V. T . (1944) . The bacteriostatic action of rose ben gal in media us ed for plate counts of soil fungi . Soil Sci. 58 , 467-471. STEPHEN, R . C. (1957). A simple spore tr ap. Nature, Lond., 179, 1360-1 361. WARCUP, J. H . ( 1950) . The soil-pla te m ethod for isola tion of soil fungi . Nature, Lond., 166, 117-1 18. WELLMAN, F . L. (196 1). Cojfee. World Crops Series, Leon ard Hill Ltd., London.
(Accepted for publication 14 December 1963)
FUNGUS FLORA OF COFFEA ARABICA IN NYASALAND By M. A. SIDDIQI Department of Agriculture, Nyasaland The fungi isolated from all parts of the plant and surroundings are listed. From six coffee sources, 81 species of fungi were isolated. The distribution of the saprophytic and pathogenic isolates is discussed.
A large amount of literature on coffee diseases exists, the most recent contribution being by Wellman (1961) in which a full survey of coffee pathology is made. While much is known about coffee diseases and their distribution, the microflora of coffee has received less attention. During a study of the bark diseases of CojJea arabica in Nyasaland during 1959 to 1962 (Siddiqi & Corbett, 1963), a record was kept of the fungi isolated from the soil, roots, and aerial parts and from a spore trap in coffee fields, and the results of this exercise are presented here. MATERIALS AND METHODS
The coffee gardens in which these observations were made are all in the coffee-growing areas of Nyasaland. Coffee soils are generally moderately acid (top soil pH 5"6-6'4) with a more acid subsoil. They are deep red sandy-clay loams usually well drained but occasionally impeded by a lateritic gravel layer at depth.
Collection of samples and isolations The numbers of samples and frequency with which they were taken from various coffee gardens were not constant because the samples were originally intended for a study of the diseases caused by a form of Fusarium stilboides Wollenw. (Siddiqi & Corbett, 1963). All isolations were made on a standard agar medium with the following composition: glucose, IO g.; KN0 3 , 1 g.; KH 2P0 4 , I g.; agar, 20 g.; rose bengal, 67 p.p.m.; tapwater, 1 1. The pH of the medium was adjusted to 6. Sometimes the standard agar medium was used without rose bengal, which was added to inhibit bacterial development (Smith & Dawson, 1944); it is very efficient in this respect and also has the advantage offavouring a more varied fungus flora than does the use of a very acid medium. The general procedure for collecting samples and making isolations (Table I) was as follows: (I) Soil. Three random samples of soil were taken to approximately 8 in. depth using a trowel. The three samples were thoroughly mixed and passed through a 2 mm. sieve. The soil was incorporated in melted agar medium (approximately 5 mg. per IO ml. medium) cooled to 50° C. and the plates poured immediately (Warcup, 1950). 18
MYC.47
Transactions British Mycological Society
282
(2) Roots. Three root samples were taken from each unhealthy plant examined. They were thoroughly washed to remove adhering soil. From these, twenty-five rootlets were selected at random and cut into small pieces. The pieces were mixed thoroughly and twenty-four pieces selected at random, surface sterilized by dipping in absolute alcohol for 30 sec., washed in sterile water and then placed on the surface of standard agar plates. (3) Bark andbranches. Pieces of infected bark and branches were surface sterilized in o· 1 % mercuric chloride solution or absolute alcohol, washed in sterile water and then placed on the surface of standard agar plates. (4) Leaves and fruit. After incubation in moist chambers these were examined directly and pieces were also plated on the standard agar medium. (5) Spore trap. A simple spore trap (Stephen, 1957) was used, placed in the middle of each coffee garden. As it was difficult to identify fungi solely from the spores on the slides by direct examination, cultures were made. Each slide was placed in a sterile Petri dish and 15 ml. standard agar medium cooled to 50° C. poured on top. Recording offungi Plates were incubated at room temperature (20-25° C.) and kept under observation for 6 weeks. Forms found difficult to identify directly by microscopic examination of the plates were isolated in pure culture. Fungi which did not sporulate are designated as unidentified species. NOTES ON THE OCCURRENCE OF THE FUNGI
From the six areas a total of eighty-one specieswas isolated and identified (Table 2). Because the isolates were originally taken for specific studies of coffee bark diseases (Siddiqi & Corbett, 1963) selective media or techniques were not used. If this had been done additional species of fungi would probably have been obtained. Table
I.
Analysis of samples, plates examined andfungi identified, August, 1959-Ju(Y, 1962 No. of samples
Bark and branches Roots Leaves Fruit Soil Spore trap
No. of plates
Saprophytic spp.
Pathogenic spp.
4
8
346
1730
100 500 300
500
78
780
54
128
128
31
4 I 2
5 9 5 4 5
The distribution of saprophytic and known pathogenic fungi is given in Table I. High numbers of saprophytic fungi occur in the soil and on spore trap slides. The few fungi isolated from roots and the aerial parts of coffee are mostly pathogenic (Table 2, P) and all are well-known coffee pathogens reported from other coffee-growing countries as well. The few saprophytes isolated from roots and aerial parts were secondary invaders.
28 3
Fungus flora of coffee. M. A. Siddiqi Table
2.
List offungi isolated from the different sources Soil
Absidia sp. Circinella simplex van Tiegh. Cunninghamella sp. Mortierella pusilla Oud. Mortierella sp. Mucor sp. M. racemosus Fresen. Rhizopus sp. Zygorrhynchus sp. Capnodium brasiliense Putten Chaetomium cochlioides Pall. Chaetomium sp. Mycosphaerella coffeae Noack Nematospora sp, Pseudogymnoascus vinaceus Raillo Thielavia terricola (Gilm. & Abb.) Emmons Westerdykella sp. Acremonium sp. Acrothecium sp. Alternaria sp. Arthrobotrys sp. Ascochyta sp Aspergillus fumigatus Fresen. A. flaous Link ex Fr. A. niger van Tiegh. Blastomyces sp. Botryotrichum sp. Botrytiscinerea Pers. ex Fr. Cephalosporium acremonium Corda Cercospora coffeicola Berk. & Cooke Cladosporium herbarum (Pers.) Link ex Fr. Cladosporium sp. Colletotrichum coffeanum Noack Curvularia sp. Cylindrocarpon sp. Diplodia sp. Fusarium solani (Mart.) Sacco F. equiseti (Corda) Sacco F. oxysporum Schlecht. ex Fries F. stilboides Wollenw. Geotrichum sp, Gliocladium roseum Bain. Helminthosporium sp. Hormiscium sp. Hormodendrum cladosporioides (Fresen.) De Vries Mycogone sp, Nigrospora sphaerica (Sacc.) Mason Oospora sp. O. egyptiaca van Beyma Papularia sp. Penicillium spp.
Bark and branches
Leaves
Fruit
+ + + + + + + + P P -
+ + +
+ +
+
+
+
+
+
+
+
P
+ + + +
P P
+ +
+
+ + + + + + +
+ +
+ + + + + + + + +
+
+
+ + + + + +
P
P P P
+
+
P
P
+ +
+ +
P
Spore trap
+ +
+ +
(5 spp.) Pestalotia sp. Phoma sp, Pullularia sp.
Roots
+ + +
+ +
+
+ +
+ + + + + + + + +
+ +
+
+
(I sp.)
+ +
(I sp.)
+
+ + +
(3 spp.)
18-2
28 4
Transactions British Mycological Society Table Soil
Sepedonium sp, Sphaeronaema sp. Spicaria violacea Abbott Thielaoiopsis sp. Ti lachlidium sp. Torula lucif uga Oud. Trichoderma viride Pers. ex Fr. T. glaucum Abbott T. koningii Oud, Trichophy ton sp. Verticillium terrestre (Link) Lindau Armillaria mellea (Vah!. ex Fr. ) Kummer Clito/iYbe sp , Hemileia vastatrix Berk, & Br. (Race II) Mycena sp. Pellicularia koleroga Cooke Rhizoctonia sp. Un identified spp.
+ + + + + + + + + +
Present .
Bark and branches
Leaves
+ +
+ +
Fruit
Spore trap
+ + +
+
(6 spp.) 58
+
Roots
+
P P
Totals
(cont).
+
P
P P P
2
+
+ + +
+ + +
9
12
10
+ +
7
(3 spp ) 36
P = pa thogenic on coffee.
The most common fungi isolate d from soil (Table 2) were Aspergillus fumigatus , A. niger, Trichoderma viride, T . koningii, Cephalosporium acremonium, Cladosporium herbarum, Cladosporium sp., Fusarium solani, F. oxy sporum, F. equiseti, Penicillium spp. and Cunninghamella sp. These, with the exception of F. oxysporum, were also common on th e spore trap slides. None of the saprophytic or pathogenic fungi was pr evalent in all the six coffee habitats studied. I wish to thank Mr D. C. M. Corbett , Department of Agriculture, Nyasaland, for helpful criticism in the preparation of this paper, the late Dr W. L. Gordon, Can ad a Dep artment of Agriculture , for confirming th e identity of Fusarium cultures, and the Director and Sta ff of th e Commonwealth Mycological Institute, K ew, for looking through th e list of fungi. This paper is published by permission of the Director of Agriculture and Fisherie s, Nyasaland. REFERENCES
SIDDIQ.I, M . A. & CORBETI, D . C. M . (1963) . Coffee bark diseases in Nyasala nd. I. Pathogeni city, description and identity of th e causal orga nism. Tr ans. B rit. mycol. Soc. 46, 9 1-IO I. S~fiTH, N. R . & DAWSON, V. T . (1944) . The bacteriostatic action of rose ben gal in media us ed for plate counts of soil fungi . Soil Sci. 58 , 467-471. STEPHEN, R . C. (1957). A simple spore tr ap. Nature, Lond., 179, 1360-1 361. WARCUP, J. H . ( 1950) . The soil-pla te m ethod for isola tion of soil fungi . Nature, Lond., 166, 117-1 18. WELLMAN, F . L. (196 1). Cojfee. World Crops Series, Leon ard Hill Ltd., London.
(Accepted for publication 14 December 1963)