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The influence of the developing groundnut fruit on soil mycoflora
[ 393 ] Trans. Br. mycol. Soc. 53 (3), 393-406 (19 69) Printed in Great Britain
THE INFLUENCE OF THE DEVELOPING GROUNDNUT FRUIT ON SOIL MYCOFLORA By D. McDONALD
Institute for Agricultural Research, Ahmadu Bello University, Samaru, Zaria, Nigeria (With 5 Text-figures) Examination offield soil and soil from around groundnut (Arachis hypogaea L.) fruits in 1965 showed that the developing fruit had both quantitative and qualitative influences upon the soil mycoflora. Further studies in the following year gave added evidence of the presence of a strong geocarposphere effect in groundnuts and emphasized the relation between certain fungi and the groundnut fruit.
It has been established by several workers (e.g. Williams & Schmittenhenner, 1960) that plant residues from the previous year's crop are more important in influencing the soil mycoflora than are exudates and sloughed off tissues from the roots of the current season's crop. However, the growing plant does have a definite influence on the soil mycoflora and this effect is greater in soil close to the roots than in soil remote from the plant. Hiltner (1904) was the first to use the term rhizosphere to denote the region in the soil surrounding the roots of a plant and affected by its secretions. Rao (1962) in India studied the rhizosphere effects of a range of groundnut varieties and found that some had strong rhizosphere effects while others had only slight effects. He also discovered that the age of the plant was of importance in determining the rhizosphere effect and that different varieties had preferential stimuli for specific fungi. Joffe & Borut (1966) investigated the soil mycoflora of groundnut fields in Israel and listed species that were consistently present. Many of these were fungi previously associated with diseases of groundut fruits and seeds. In an experiment carried out at Samaru, Nigeria, Dransfield & McDonald (1966) investigated the effects of cropping practice and manurial treatment on the soil mycoflora using groundnuts, sorghum and cotton as test plants. They found that the different crops exerted little total quantitative effect but did stimulate some fungal species preferentially. If the groundnut fruit were to possess a selective influence upon the soil mycoflora with which it is in close contact during development this might explain the close similarities of mycofloras of shells and seeds of crops grown in different soils in different parts of the world. To investigate this possibility, experiments were carried out during the 1965 and 1966 growing seasons at Mokwa Agricultural Research Station, Nigeria. In the first year mycofloras of soil remote from plants and of soil from around developing fruits were examined, while in the second year the geocarpo-
394
Transactions British Mycological Society
sphere mycoflora only was studied. The geocarposphere is a term coined by Garren (1966) to denote the zone of enhanced microbiological activity in the soil immediately adjacent to the hypogeous fruit. MATERIALS AND METHODS
The long-season variety Samaru 38 (Castle Cary Bunch Group) was used in both years, seeds being sown at 23 cm spacing along ridges 91.4 em apart. Sowing dates were 2 June 1965 and 4 May 1966. Mokwa (9° 19' N, 5° 00' E) has an annual rainfall of 1000-1270 mm distributed largely between 1 Apr. and 31 Oct. The soil is classified as a red ferrisol on loose sandy sediments, and the Dangappe series on which the experiments were sited is characterized by its freedom from gravel, stones, concretions and mottling and by its great soil depth.
Soil sampling In both experiments the crop was laid out in twenty replicate plots to facilitate sampling. In 1965, samples of soil from around fruits (fruit soil) were first taken 7 weeks after sowing and further samples taken at weekly intervals until harvest. Samples of soil from between plants (field soil) were collected at the same times. In 1966 the geocarposphere soil was first sampled 9 weeks after sowing and then at weekly intervals until harvest. Methods of sampling are described below. Field soil (1965). Using a sharp knife, a vertical cut was made in the ridge to a depth of 15 cm and parallel to it in a position midway between two plants. A sterilized metal tube of 2 em internal diameter was pushed into the soil at right angles to the cut face and to a depth of 10 cm. Two sample cores were taken from each plot and the resulting forty samples were bulked and mixed thorougWy. The composite sample was used for investigation of the mycoflora. Fruit soil (1965). One plant was taken from each plot, shaken to remove loosely adhering soil particles, and the pods showing most advanced development were detached. These pods were then placed in a clean polythene bag and shaken vigorously for 5 min to dislodge all but the most firmly adhering soil. The soil removed in this way was bulked, thoroughly mixed, and the resulting composite sample was used in the mycoflora investigations. Geocarposphere soil (1966). The main difference between fruit soil sampled in 1965 and geocarposphere soil sampled in 1966 was that the latter included soil particles which were in close contact with the fruit surface and which were not detached from pods receiving the type of treatment used in collection of fruit soil in 1965. To obtain geocarposphere soil, one plant was taken from each plot, shaken to remove loosely adhering soil, and the most mature pods detached. The pods taken from each plant were bulked and from them were selected four random samples of ten undamaged, two-seeded pods. Each lot of ten pods was placed in a flask containing 50 ml of sterile, distilled water. Sterilized rubber bungs were fitted to the flasks, which were then shaken
Soil mycoflora. D. McDonald
395
for 2 min at 250 oscillations per minute. The resulting soil suspension was examined by the dilution plate technique for numbers and species offungi present. Examination of soil mycofloras Field and fruit soils were examined by the dilution plate technique and by the Warcup soil plate method (Warcup, 1950). The geocarposphere soil was examined by the dilution plate technique only. In the 1965 experiment the initial soil suspension used in preparing the dilution series was made by adding about 4 g soil to 100 ml sterile, distilled water in a 250 ml flask. Dilutions of I: 1000 and I: 10000 were selected for the tests. Five replicate dilution series were prepared and two plates made from the selected dilutions of each replicate set. The weight of dry soil in the original sample used in the preparation of the basic soil suspension was found by evaporating the water and weighing the soil remaining, the small quantities removed when making the dilution series being neglected. In the 1966 experiment, four initial soil suspensions were obtained as described earlier. Dilutions of I: 5000 and I: 50000 were selected for test and two plates were prepared from each dilution. Dilution plates were prepared by the addition of 1 ml of the selected soil suspension to a sterile, 9 cm diam Petri dish to which was added some 20 ml of cooled, molten Czapek Dox-rose bengal-streptomycin agar (CDRBSA). The dish was rotated to mix suspension and medium. The CDRBSA medium was made by adding to standard Czapek Dox agar sufficient rose bengal to give a concentration in the medium of 1 part in 20000 and adding streptomycin sulphate to give a concentration of 50 pg per ml of medium. The antibiotic was added to the medium after cooling and shortly before the plates were poured. Plates were incubated at room temperature (27-32 0c) for 5-6 days and fungal colonies that developed were counted and identified. Each colony was considered to have originated from one fungal propagule. Unknown species were sub-cultured on to potato dextrose agar and Czapek Dox agar slants for further study. Ten Warcup plates were made from each offield and fruit soils at each time of sampling. The standard method was used and the medium employed was corn meal agar, 15 ml of the cooled, molten medium being added to each dish. The plates were incubated at room temperature and checked for species of fungi present after 7 days and again after 14 days. In both experiments the moisture content of field soil was determined at each time of sampling. Soil samples were weighed, dried for 5 h at 105°, the weight of water evaporated found and expressed as percentage of wet weight, RESULTS
In both experiments the moisture content of field soil fluctuated considerably from week to week, In 1965, the range of moisture content observed was 4'1-11'2 %, while in 1966 it was 5'9-12'3 %. Both experiments were conducted during the rains and no obvious effects of soil moisture upon fungal numbers were evident in either year.
Transactions British Mycological Society Table
Mean numbers of active fungal propagules per g of dry soil infield,fruit, and geocarposphere soils
I,
.
Numbers (thousands) Weeks from sowing 8 9
22
46 48
12
29
10
21
II
19 19
39
12
12
13
27
42
7
15
16 17 18
Geocarposphere soil
76 80 51 91
47 74 88
18 9° 34
14
,
Fruit soil
Field soil 16
139 227 327
97
145 344
176
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•
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14
15
16
17
18
Weeks from sowing Fig.
I.
Geocarposphere soil: numbers of fungal propagules and mean weights of soil per ten-pod sample.
In Table I times ofsampling are presented in terms ofweeks from sowing and numbers of active fungal propagules per gram of dry soil are given for field, fruit and geocarposphere soils. The numbers are based on average plate counts; from the I: 1000 dilutions in the case offield and fruit soils, and from the I :5000 dilution for geocarposphere soil. Weights of soil used in preparation of the dilution series were reasonably constant in 1965, but in the 1966 experiment the weights of soil adhering to the lo-pod samples
Soil mycofiora.D. McDonald
397
varied considerably from week to week and this affected the quantitative results obtained. For geocarposphere soil numbers ofpropagules and mean weights of soil adhering to ten-pod samples are shown in Fig. I. All fungi isolated are listed in Table 2. Results of Warcup plate tests are given for selected fungi in Table 3. The results of the dilution plate tests are presented in Table 4 for field, fruit and geocarposphere soils, the figures shown being percentages of total fungi present at each sampling occasion made up by each species. Numbers of active propagules present in each soil environment are given in Table 5 for a few selected species. 100 ;:;-0 .....
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15
16
Weeks from sowing
Fig.
2.
Numbers offungal propagules per g dry soil. e - e , Field soil; 0- - -0, fruit soil.
From consideration of Table 1 and Fig. 2 it is evident that the fruit soil contained higher numbers of fungal propagules than did the field soil on all but one sampling date. On the single occasion (week 15) when numbers of fungi in field soil were about the same as numbers in fruit soil, it was noted that an unusually high number ofpropagules of Aspergillus flavus was recorded from the field soil. As field soil examined on the following week contained much fewer propagules of A. flavus, it was thought that the sample taken on week 15 was atypical, containing numbers of conidia of A. flavus out of all proportion to the vegetative activity of the fungus in the soil at that time. Total fungi showed a steady increase in numbers in fruit soil from week 10 to week 16, but there was only a slight increase in fungal numbers in field soil during the latter part of the sampling period (Fig. 2). Examination of geocarposphere soil showed that numbers of active fungal propagules per g of dry soil varied inversely with the weight of soil adhering to the ro-pod sample (Fig. I). However, the regression coefficient in the analysis of covariance was not significant and no adjustment
39 8
Transactions British Mycological Society Table
2.
List offungi isolatedfromfield (F),fruit (FR), and geocarposphere (G) soils Warcup plates ~
Absidia sp. Acremonium sp. Alternaria spp. Arthrobotrys sp. Aspergillus caluJidus Link A. carneus (Tiegh.) Bloch A.j/auipes (Bain. & Sart.) Thorn & Church A.flavus Link ex Fr. A. fumigatus Fres. A. glaucus Link A. nidulans (Eidarn) Wint. A. niger v. Tiegh. A. oehraceus Wilh. A. tamani Kita A. terreus Thorn A. ustus (Bain.) Thorn & Church A. versicolor (Vuill.) Tirab. Bahusandhika caligans Batista & Upadh. Botryodiplodia theobromae Pat. Botrytis sp. Cephaksporium acremonium Corda C. roseo-griseum Sabena C. spp. Chaetomium atrobrunneum Ames C. seminudum Ames C. spp. Circinella sp. Cladosporium herbarum (Pers.) Link CWl1IinghaT1ll!lla elegans !.encin. Curvularia lunata (Wakk.) Baed. C. spp. Cylindroearpon sp. Fusarium spp. Glioeladium sp. Gonatobotrys sp. Helminthosponum spp. Hendersonula toruloitka Nattr. Hormodendrum sp. Humicola fuseoatra Traaen H. fusisporus Traaen H. grisea Traaen Macrophomina phaseoli (Maubl.) Ashby Monilia sp. Monotospora sp. Mortierella sp. Mucor spp. Myrotheeium indicum Rarna Rao M. verruearia (Alb. & Schw.) Ditrn. Neocosmospora africana. Arx N. vasinfteta E. F. Sm. Nigrospora sp. Oospora sp. PaeciWmyces fusisporus Saks. P. varioti Bain. P. spp. Pellicularia sp. Penicillium citrinum Thorn P. ehrlichii Kleb. P.frequmtans Westling
F
FR
X
X
x
x
Dilution plates A
F
FR
G X
X X
x
x
X
X
x x
x x x
x x x
x x x
x x
x x
x x x x
x x x
x x
x x x
x x x
x x x x
x
x
x
x
x x x x
X
x x x
x
x x x x
x x x
x x x
x x x
x x x
x x x x x x
x
x
x
x
x x x x
x x
x x x x
x x
x x
x x
x x
x x x
x
X
x x
x x x x
x x x
x
x
x
x
x x
x
x
x x x x x
x x x x
x x x x x
Soil mycofiora. D. McDonald Table
2
399
(cant.) Dilution plates
Warcup plates ~
F
P. funiculosum Thorn P. islandicum Thorn P. variabile Sopp. P. spp. Peyronellaea glomerata (Corda) Goidimich Plwma eupyrena Sacco P. prunicola (Opiz) Wr. & Hochapf P. spp. Pullularia pullulans (de Bary) Berkhout Pyrenochaeta sp. Rhizoctonia solani Kuehn Rhizopus arrhizus Fisch. R. stolonifer (Ehrenb. ex Fr.) Lind. R. spp. Sclerotium roifsii Sacco Spicaria sp. Stachybotrys atra Corda S. spp. Stemphylium sp. Syncephalastrum racemosum (Cohn.) Schroet. S. spp. Tetracoccosporium paxianum Szabo Thielavia basicola Zopf. Torula sp. T riclwikrma oviferum Rifai T. viride Pers. ex S. F. Gray Verticillium spp.
FR
A (
FR
G
x
x
x
x
x
x
x x
x x
x
x
x
x
x
x
x
x
x
x
x
x
F
x x x x x x x x x x x x x x
x x
x
x x
x x
x x x x
x x x x x
x x
x
x x
45
39
27
33
51
Table 3. Warcup plate tests. Numbers ofplates (flO) on which fungi were present Weeks from sowing
,
A
Aspergillus jlavus Botryodiplodia theobromae Fusarium spp. Macrophomina phaseoli Penicillium spp. Rhizoctonia solani Trichoderma viride
Soil F FR F FR F FR F FR F FR F FR F FR
7
8
9
10
II
12
13
14 I
I
I
I
1 10 8
3 3 5 4 2 9 10
2 4 9 8 4 6 10 9
0 6 5 8 4 10 10
3 6 7 9 3 5 9 10
3 8 7 7 2 5 10 10
3 8 6 9 2 9 10 10
3 7 7 10 2 4 10 10
2 9 9 10 2 8 10 9
3
2 3
3
2 3
I
I
3 10 10
15
16
3 7 9 10 3 7 10 10
I
3 4 2
2
3 2
I
5 2
3 2 3
Totals (/100) 0 3 21 59 72 84 24 53 98 98 1 II
27 14
F-field soil; FR-fruit soil.
was made to the results. The high numbers of fungal propagules present in the geocarposphere at weeks 15 and 17 reflected the presence of large numbers of active propagules of Penicillium funiculosum and A. flavus respectively.
Table 4, Dilution plate tests, Fungi expressed as percentages of total isolated Field soil. Weeks from sowing
.
, 8
7 Aspergillus cameus A·fiavipes A·fiavus A·fumigatus A. nidulans A. niger A. ochraceus A. tamarii A. ustus A. versicolor Bahusandhika caligans Botryodiplodia theobromae Cephalosporium spp. Chaetomium spp. Cunninghamella elegans Curvularia spp. Fusarium spp. Hendersonula toruloidea Humicola spp. Macrophomina phaseoli Myrothecium spp. Neocosmospora africana No vasinfecta Paecilomyces spp. Penicillium citrinum P. funiculosum P. spp. Phoma spp. Rhizoctonifl solani Rhizopus spp. Stachybotrys spp. Tetracoccosporium paxianum Thielavia basicola Trichoderma spp.
-
-
-
-
2
I
6 6 13 3
37 8 12
-
9
-
-
10
-
-
3
II
16 -
5 15 12 9 I
12
13
14
15
16
-
-
-
-
-
2
2 6
4 6 16
I
-
10 15 8 4 -
-
8 10 -
-
-
-
-
5 3 3 2 2 3 3 4 - - - - - 2 - -
-
-
-
26
-
15
-
-
-
-
-
-
13
4 I
-
-
-
-
-
I
36 -
-
-
-
-
-
-
-
I
II
I
-
-
53
-
-
-
-
-
-
-
48 -
I
-
I
-
-
-
4
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I
-
-
-
-
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2 2
2 -
-
2
-
.
t
-
I I
3 13 17
2
II
-
-
-
-
4
2 2 2
- - I -
- - 2 I 6 3 - - - I - I 63
-
-
2 8
-
10
-
2
34
-
-
I
-
6
-
I
-
-
-
-
7 6 6 7
I
-
2
-
2 28 2 2
-
13
14
15
16
-
-
-
-
-
-
-
-
2 5 8 3
2 3
28
-
2 3
I
-
12
I
I I
II
-
-
10
I I
-
I
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10 2
3 2 4
7 4 3
4 9 7 2
I
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I
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6
3
2
-
-
8 2
-
-
5 3
-
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-
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36
35
36
33
-19
-
II
-
14 4
-
I
I
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22
10
5 2
-
I I I
-
-
2
-
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10 5
4 2 4 4
2
2
22 4
10 2 3
-
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I -
I
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-
-
6
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2 3
43
33
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24 4
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-
-
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2 20 9
3 2
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+
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7
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22
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17
18
+ + +
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17 3 I
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25
14
-
-
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+ + 4
+
-
9 II
-
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6
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4
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-
The percentages given in the above table are to the nearest whole number; figures of less than 0'5 % are shown as
3 20
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3 24 3
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2 30 4
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2 16 2
3 6 3 3
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5 7
5 5
10
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5 8 -
8
,
A
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4 2
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Geocarposphere soil. Weeks from sowing
I
-
2 - -
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2
2
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-
2
8
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,
10
-
-
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3
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4 13
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34 38 2 5 - -
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79
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6 2 2
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4 3 2 3
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56 63 -
-
-
8 5
2
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-
2 3 2 2 8
-
-
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29 2 2 2
7
-
-
II
,
Fruit soil. Weeks from sowing
9 I
+ +
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Soil mycoflora. D. McDonald
401
The list of species (Table 2) illustrates the influence of the isolation technique and culture medium used on the range offungal species isolated, a wider range of species being isolated from both field and fruit soils by the Warcup soil plate method than by the dilution plate technique. Irrespective of isolation technique, totals of 57, 54 and 52 species were found in field, fruit and geocarposphere soils respectively. Of the fifty-two species from geocarposphere soil, twenty-five had been found in fruit soil in the previous year and of these twenty-four had also been isolated from Table 5. Numbers in thousands of active fungal propagules per g of dry soil in field (F),fruit (FR) and geocarposphere (G) soils Weeks from sowing
,
A
7
Aspergillus spp,
F 4'5 FR 2,8 G 0 Aspergillus F 0'2 jiavus FR 0'7 G 0 Aspergillus F 0'9 fumigatus FR 0'9 G 0 Botryodiplodia F 0 theobromae FR 0 G 0 Fusarium F 4'0 spp, FR 15'6 G 0 Macrophomina F 0 FR 0 phaseoli G 0 Penicillium F 0 spp, FR 3'4 G 0 Rhizocfonia F 0 solani FR 0 G 0
8 12'7 10'7 0 0'2 1'9 0 8'2 0'5 0 0 0 0 6'3 17'9 0 0'4 0 0 0 13'8 0 0'2 0 0
9 3,6 7'3 17'0 0'4 2'0 2'7 0 1,6 7'1 0 0 0 6'4 8'2 22'7 0 0'4 9'4 1'3 6'4 3'7 0 0'4 0
10
II
12
13
14
15
16
17
7'9 3'7 19,8
7'0 5'0 15'0 0 0'4 0'4 1,8 0,6 8'7 0 6'2 0
3'3 2'7 14'3 0 0 2'3 0'4 0'2 5'3 0 3'4 11'2
7'1 4'2 47'1 1'5 0'2 2'1 0'3 0'5 21'9 0'5 2'4 6,6
2'7 11'1 72'4 0'5 7'0 7'5 0'3 1'5 52'0 0 4,8 2,6
14'4 14'5 53'4 0'9 6'8 12,8
0 0 199'9 0 0 10 7'8 0 0 24'2 0 0 3,8
9'1 13'9 12'9 0 5'2 3'5 0,8 2'5 3'1 0 0 0
6'9 14'5 36 '2 0'4 8'4 10'3 0'2 3'1 4'1 0 0'4 0'1
16'9 16'8 44'0 0 4'5 5'5 0 12'3 14'7 0 0 1'1
77'1 19'4 23'9 70 '8 2,6 0'5 0'5 1,8 15'7 0 4,8 1'9 8,6 37'S 47'2 1'4 4'4 6'8
11'4 24'0 56 '2 0'3 17,8 23'2 1'1 0'5 8'5 8'4 6,8 224'4 0 0 0 0 0 0'2
0'9 0'4 0'2 2'9 1'1 13,8 0'2 0'4 0 6,8 4'7 19'3 0'2 4'0 4'2 0 2'2 20'2 0 0'2 0
4'2 3'9 17'5 0 1'4 3'1 11'4 3 1'9 28'0 0 4'8 4'9 2'5 29'S 21'7 0'3 0'5 1'4
18
0 0 68'2 0 0 4 2'2 0 0 16'3 0 0 2'0 0 0 0 0 56 '1 58 '8 0 0 0 0 19'4 10'2 0 0 0 0 16'0 16,6 0 0 0 0 0 0
field soil. Species restricted to anyone specific soil environment were not of common occurrence and were not abundant at any time, Some of the more important and more frequently occurring fungi are considered below. The percentage of aspergilli of total fungi varied through the sampling period in both field and fruit soils, In general, the fungi were more abundant and made up higher percentages of total fungi in field than in fruit soil. In the geocarposphere, Aspergillus spp. were dominant on six occasions and on only one time of sampling (week 15) did they fall as low as third position. On Warcup plates the genus was well represented with A. niger and A. fumigatus the most abundant. The percentage of A. flavus in the mycoflora fluctuated throughout the sampling period in both field and fruit soils, but in both soils the highest figures were recorded during weeks 14-16. In the geocarposphere, A. 26
Myc, 53
402
Transactions British Mycological Society
jlavus showed highest levels, in terms of both numbers of propagules and percentages of total fungi, during weeks 16-18. A. jlavus was not found on Warcup plates from field soil but was present on those from fruit soil sampled at weeks 14-16. A. fumigatus was of frequent occurrence in all soil environments, but was more abundant in field than in fruit soil. In the geocarposphere A.fumigatus composed high percentages oftotal aspergilli during weeks 9- 15. A. nidulans composed higher percentages of total fungi in field than in fruit soil. It was more frequent in Warcup plates from field soil than in those from fruit soil. In geocarposphere soil, A. nidulans had its highest percentages on weeks 16 and 17. A. niger was a common species in all soil environments, being present in higher proportions in field than in fruit soil. It was common at all times on Warcup plates prepared from both field and fruit soils. The fungus was present in geocarposphere soil at all times of sampling. A. ochraceus was not of common occurrence in field or fruit soils but was isolated on weeks 7 and 10 in the former and on week 7 in the latter environment. The fungus was not found on Warcup plates. In geocarposphere soil, A. ochraceus was present on all but one sampling occasion but never composed a high proportion of total fungi. Botryodiplodia theobromae was found on weeks 10 and 13 in field soil and on weeks 10-16 in fruit soil. This fungus was more abundant and composed higher percentages of total fungi of fruit than of field soil. In Warcup plate tests B. theobromae was also isolated more often from fruit than from field soil and showed a tendency to increase in abundance as the fruit developed. In the geocarposphere the fungus was not isolated until week 12, but was found on all subsequent samplings. The various isolates of fusaria obtained in these investigations were not identified to species. The fungi were common in all soil environments. As a percentage of total fungi Fusarium spp. were more important in field soil than in fruit soil, but in terms of numbers of active propagules present the reverse was true. In the geocarposphere the fusaria increased in numbers as the fruit developed, but tended to form a lower proportion of the total mycoflora. Macrophomina phaseoli (syn. Sclerotium bataticola) was uncommon in field soil but was generally abundant in fruit soil, where it composed appreciable percentages of the total fungi, as was shown in both dilution and Warcup plates. M. phaseoli increased in numbers of propagules in the geocarposphere as the fruit matured but there was a tendency for its percentage of total fungi to decrease with time although a slight increase was manifest during the last three times of sampling. Penicillium spp. were present in greater numbers and composed higher percentages of total fungi in fruit than in field soil. In the geocarposphere the proportion of Penicillium spp. in the mycoflora increased slightly as the fruit developed, but numbers of propagules increased to a considerably greater degree. The species most frequently isolated were P. citrinum and P. funiculosum. Rhizoctonia solani was present in all soil environments but not common in any. It occurred rather more often in fruit than in field soil.
Soil mycojWra. D. McDonald
403
Trichoderma viride, despite being isolated from field and fruit soils by both dilution plate and Warcup plate techniques, was of very infrequent occurrence. T. viride was not found in geocarposphere soil in 1966, but the related species T. oviferum was isolated on one occasion. DISCUSSION AND CONCLUSIONS
The 1965 experiment showed that the developing fruit had a strong influence on the mycoflora of the soil surrounding it, numbers of fungal propagules being much higher in fruit than in field soil. The I9~6 investigations indicated an increase in numbers of fungal propagules ill the 16 15
14 l>O
c:
'i
13
0
'" 12
E
...
0
11
I II
~
1lI 1lI
~
9
8 7 Fig. 3. Field soil, selected fungi as percentages of total isolates. Key: see Fig. 5.
geocarposphere as the fruit developed. The quantitites of soil adhering to the ten-pod samples fluctuated considerably from week to week, being influenced by soil moisture content and stage of development of the fruit. It is possible that variation in quantity and nature of exudates from the fruit might influence the adherence of soil, either directly or indirectly by affecting fungal and bacterial growth leading to binding together of soil particles. Numbers of fungal propagules estimated per gram of dry soil varied inversely with the amount of soil adhering to the ten-pod sample and this was almost certainly due to diminution of the geocarposphere effect in soil remote from the fruit surface. The percentages of total fungi in field, fruit and geocarposphere soils composed by four important groups of fungi are shown in Figs. 3,4 and 5 respectively. It is evident that the dominant fungi in all three soil environments were Fusarium spp. and Aspergillus spp., the former being predominant in field and fruit soils and the latter in geocarposphere soil. Penicillium spp. and Macrophomina phaseoli were important in fruit and geocarposphere soils but less significant in field soil. Other fungi which were more abundant in fruit and geocarposphere soils than in field soil include 26-2
404
Transactions British Mycological Society
Botryodiplodia theobromae, Myrothecium verrucaria, Phoma spp. and Rhizoctonia solani. The above results differ from those obtained in Georgia, U.S.A., by Jackson (1968) when he investigated the mycoflora of soil washed from the surfaces of groundnut fruits. He sampled the soil from fruits lifted at intervals over a period of 6 weeks leading up to harvest and found that the 16 15 14
~
c
'i
13
0 on
E 12
...0
on
.,:,t. Q) Q)
~
11 10 9 8 7 Fig. 4. Fruit soil, selected fungi as percentages of total isolates. Key: see Fig. 5.
18
17 16 DO
.=~
15
on
14
~
13
o
E o
11 10
f}:;:J Aspergillus spp.
~PeniCillium spp.
I:: :1 Fusarium spp.
•
D
Other fungi
Macrophomina phaseoli
Fig. 5. Geocarposphere soil, selected fungi as percentages of total isolates.
Soil mycoftora. D. McDonaid dominant fungi were, in order of abundance, Penicillium spp., Aspergillus spp. and Fusarium spp. The reason for the different order is unknown but could be due to different soil conditions during fruit maturation. The balance of the penicillia and aspergilli could be influenced by soil temperature, lower temperatures being likely to favour the former. Many fungi were common to fruit and geocarposphere mycofloras in Nigeria and most of these are included in the list compiled by Jackson (1965) of fungi reported on groundnut pods and kernels. A considerable number of the fungi isolated in Nigeria have also been isolated in Israel by Joffe & Borut (1966), who investigated the mycoflora of groundnut fields in different parts of the country. Two interesting species found in Mokwa soil, but not isolated in Israel, are the toxigenic Penicillium citrinum and Aspergillus ochraceus. Most of the aspergilli and penicillia listed in Table 2 are known to be capable of producing toxic metabolites. It is of interest to note that Fusarium spp. have on many occasions been isolated from rotting pods and seeds, Botryodiplodia theobromae and Macrophomina phaseoli are common causes of concealed damage of seeds, and Rhizoctonia solani is a pathogen of roots, pegs and pods. If soil associated with diseased pods were to be examined instead of soil from around undamaged pods, it is likely that the proportions of the more parasitic species in the mycoflora would be much increased. In conclusion, although the techniques used ostensibly influenced the constitution of the mycofloras of field and fruit soils, quantitative and qualitative influences of the fruit upon the fungi in the soil around it were apparent. Numbers of fungal propagules were consistently higher in fruit soil than in field soil and Botryodiplodia theobromae, Macrophomina phaseoli, Myrothecium verrucaria, Penicillium spp., Phoma spp. and Rhizoctonia solani were all stimulated preferentially by the developing fruit. Geocarposphere soil was similar in species composition to fruit soil, but contained higher numbers of active fungal propagules. The presence of an inverse relation between weights of soil sampled and numbers of fungal propagules present in the geocarposphere gave added evidence for the presence of a strong geocarposphere effect. Thanks are due to the Director, Institute for Agricultural Research, for permission to publish this paper. I wish to express my appreciation to Dr Eva R. Sansome, a.B.E., and to Professor Margaret A. Keay for advice and encouragement during this work. Thanks are also due to the Commonwealth Mycological Institute for identification of a large number of fungal isolates. REFERENCES
DRANSFIELD, M. & McDoNALD, D. (1966). The influence of cropping practice and application offarmyard manure on the soil microflora at Samaru, Northern Nigeria. Niger. agric. ]nl 3, 42-55. GARREN, K. H. (1966). Peanut (groundnut) microfloras and pathogenesis in peanut pod rot. Phytopath. Z. 55, 359-367. HILTNER, L. (1904). Uber neuere Erfahrungen und Probleme auf dem Gebiet der Bodenbakteriologie und unter besonderer Beriicksichtigung der Griindiingung und Brache. Arb. dt. LandwGes. ga, 59-78.
Transactions British Mycological Society C. R. (1965). A list of fungi reported on peanut pods and kernels. Mimeogr. Ser Ga Coastal Plain Exp. Stn N.S. 234. JACKSON, C. R. (1968). A field study of fungal associations on peanut fruit. Res. Bull. Univ. Ga agric. Exp. Stn no. 26. JOFFE, A. Z. & BoRUT, SmRAY (1966). Soil and kernel mycoflora of groundnut B.elds in Israel. Mycologia 58, 62g-640. RAo, A. S. (1962). Fungal populations in the rhizosphere of peanut (Arachis hypogaea L.). Pl. Soil.7, 2, 260-266. WARCUP, J. H. (1950). The soil-plate method for isolation of fungi from soil. Nature, Lond. • 66, II? WILLIAMS, L. E. & SCHMITTENHENNER, A. F. (1960). Effect of growing crops and crop residues on soil fungi and seedling blights. Phytopathology 50, 22-25. JACKSON,
(Accepted for publication
22
June 1969)
THE INFLUENCE OF THE DEVELOPING GROUNDNUT FRUIT ON SOIL MYCOFLORA By D. McDONALD
Institute for Agricultural Research, Ahmadu Bello University, Samaru, Zaria, Nigeria (With 5 Text-figures) Examination offield soil and soil from around groundnut (Arachis hypogaea L.) fruits in 1965 showed that the developing fruit had both quantitative and qualitative influences upon the soil mycoflora. Further studies in the following year gave added evidence of the presence of a strong geocarposphere effect in groundnuts and emphasized the relation between certain fungi and the groundnut fruit.
It has been established by several workers (e.g. Williams & Schmittenhenner, 1960) that plant residues from the previous year's crop are more important in influencing the soil mycoflora than are exudates and sloughed off tissues from the roots of the current season's crop. However, the growing plant does have a definite influence on the soil mycoflora and this effect is greater in soil close to the roots than in soil remote from the plant. Hiltner (1904) was the first to use the term rhizosphere to denote the region in the soil surrounding the roots of a plant and affected by its secretions. Rao (1962) in India studied the rhizosphere effects of a range of groundnut varieties and found that some had strong rhizosphere effects while others had only slight effects. He also discovered that the age of the plant was of importance in determining the rhizosphere effect and that different varieties had preferential stimuli for specific fungi. Joffe & Borut (1966) investigated the soil mycoflora of groundnut fields in Israel and listed species that were consistently present. Many of these were fungi previously associated with diseases of groundut fruits and seeds. In an experiment carried out at Samaru, Nigeria, Dransfield & McDonald (1966) investigated the effects of cropping practice and manurial treatment on the soil mycoflora using groundnuts, sorghum and cotton as test plants. They found that the different crops exerted little total quantitative effect but did stimulate some fungal species preferentially. If the groundnut fruit were to possess a selective influence upon the soil mycoflora with which it is in close contact during development this might explain the close similarities of mycofloras of shells and seeds of crops grown in different soils in different parts of the world. To investigate this possibility, experiments were carried out during the 1965 and 1966 growing seasons at Mokwa Agricultural Research Station, Nigeria. In the first year mycofloras of soil remote from plants and of soil from around developing fruits were examined, while in the second year the geocarpo-
394
Transactions British Mycological Society
sphere mycoflora only was studied. The geocarposphere is a term coined by Garren (1966) to denote the zone of enhanced microbiological activity in the soil immediately adjacent to the hypogeous fruit. MATERIALS AND METHODS
The long-season variety Samaru 38 (Castle Cary Bunch Group) was used in both years, seeds being sown at 23 cm spacing along ridges 91.4 em apart. Sowing dates were 2 June 1965 and 4 May 1966. Mokwa (9° 19' N, 5° 00' E) has an annual rainfall of 1000-1270 mm distributed largely between 1 Apr. and 31 Oct. The soil is classified as a red ferrisol on loose sandy sediments, and the Dangappe series on which the experiments were sited is characterized by its freedom from gravel, stones, concretions and mottling and by its great soil depth.
Soil sampling In both experiments the crop was laid out in twenty replicate plots to facilitate sampling. In 1965, samples of soil from around fruits (fruit soil) were first taken 7 weeks after sowing and further samples taken at weekly intervals until harvest. Samples of soil from between plants (field soil) were collected at the same times. In 1966 the geocarposphere soil was first sampled 9 weeks after sowing and then at weekly intervals until harvest. Methods of sampling are described below. Field soil (1965). Using a sharp knife, a vertical cut was made in the ridge to a depth of 15 cm and parallel to it in a position midway between two plants. A sterilized metal tube of 2 em internal diameter was pushed into the soil at right angles to the cut face and to a depth of 10 cm. Two sample cores were taken from each plot and the resulting forty samples were bulked and mixed thorougWy. The composite sample was used for investigation of the mycoflora. Fruit soil (1965). One plant was taken from each plot, shaken to remove loosely adhering soil particles, and the pods showing most advanced development were detached. These pods were then placed in a clean polythene bag and shaken vigorously for 5 min to dislodge all but the most firmly adhering soil. The soil removed in this way was bulked, thoroughly mixed, and the resulting composite sample was used in the mycoflora investigations. Geocarposphere soil (1966). The main difference between fruit soil sampled in 1965 and geocarposphere soil sampled in 1966 was that the latter included soil particles which were in close contact with the fruit surface and which were not detached from pods receiving the type of treatment used in collection of fruit soil in 1965. To obtain geocarposphere soil, one plant was taken from each plot, shaken to remove loosely adhering soil, and the most mature pods detached. The pods taken from each plant were bulked and from them were selected four random samples of ten undamaged, two-seeded pods. Each lot of ten pods was placed in a flask containing 50 ml of sterile, distilled water. Sterilized rubber bungs were fitted to the flasks, which were then shaken
Soil mycoflora. D. McDonald
395
for 2 min at 250 oscillations per minute. The resulting soil suspension was examined by the dilution plate technique for numbers and species offungi present. Examination of soil mycofloras Field and fruit soils were examined by the dilution plate technique and by the Warcup soil plate method (Warcup, 1950). The geocarposphere soil was examined by the dilution plate technique only. In the 1965 experiment the initial soil suspension used in preparing the dilution series was made by adding about 4 g soil to 100 ml sterile, distilled water in a 250 ml flask. Dilutions of I: 1000 and I: 10000 were selected for the tests. Five replicate dilution series were prepared and two plates made from the selected dilutions of each replicate set. The weight of dry soil in the original sample used in the preparation of the basic soil suspension was found by evaporating the water and weighing the soil remaining, the small quantities removed when making the dilution series being neglected. In the 1966 experiment, four initial soil suspensions were obtained as described earlier. Dilutions of I: 5000 and I: 50000 were selected for test and two plates were prepared from each dilution. Dilution plates were prepared by the addition of 1 ml of the selected soil suspension to a sterile, 9 cm diam Petri dish to which was added some 20 ml of cooled, molten Czapek Dox-rose bengal-streptomycin agar (CDRBSA). The dish was rotated to mix suspension and medium. The CDRBSA medium was made by adding to standard Czapek Dox agar sufficient rose bengal to give a concentration in the medium of 1 part in 20000 and adding streptomycin sulphate to give a concentration of 50 pg per ml of medium. The antibiotic was added to the medium after cooling and shortly before the plates were poured. Plates were incubated at room temperature (27-32 0c) for 5-6 days and fungal colonies that developed were counted and identified. Each colony was considered to have originated from one fungal propagule. Unknown species were sub-cultured on to potato dextrose agar and Czapek Dox agar slants for further study. Ten Warcup plates were made from each offield and fruit soils at each time of sampling. The standard method was used and the medium employed was corn meal agar, 15 ml of the cooled, molten medium being added to each dish. The plates were incubated at room temperature and checked for species of fungi present after 7 days and again after 14 days. In both experiments the moisture content of field soil was determined at each time of sampling. Soil samples were weighed, dried for 5 h at 105°, the weight of water evaporated found and expressed as percentage of wet weight, RESULTS
In both experiments the moisture content of field soil fluctuated considerably from week to week, In 1965, the range of moisture content observed was 4'1-11'2 %, while in 1966 it was 5'9-12'3 %. Both experiments were conducted during the rains and no obvious effects of soil moisture upon fungal numbers were evident in either year.
Transactions British Mycological Society Table
Mean numbers of active fungal propagules per g of dry soil infield,fruit, and geocarposphere soils
I,
.
Numbers (thousands) Weeks from sowing 8 9
22
46 48
12
29
10
21
II
19 19
39
12
12
13
27
42
7
15
16 17 18
Geocarposphere soil
76 80 51 91
47 74 88
18 9° 34
14
,
Fruit soil
Field soil 16
139 227 327
97
145 344
176
•,I
•
,I I I
~ 300 I-
, I
I
I I I I
I
'
I
I
'\ I
,\
I I
I
I •
~
b4
.;!
'0 Ql
"
E
/ 1001-1
I
I
I
'.
I
,
I
, , I I,
\,
,
'
•
\\
,
~
Z
.
,"
'-
,
, I
:
I\ ,I
I"
,
200 I-
c:
.... 3 '
\
: ,
\
I'
\
2
,
I
:3 on "a.0 0
...
~
Ql
a.
,\'I
.
.,/"
·1----·1......", ....' " " 9
I
I
10
11
".
12
I
I
I
I
13
14
15
16
17
18
Weeks from sowing Fig.
I.
Geocarposphere soil: numbers of fungal propagules and mean weights of soil per ten-pod sample.
In Table I times ofsampling are presented in terms ofweeks from sowing and numbers of active fungal propagules per gram of dry soil are given for field, fruit and geocarposphere soils. The numbers are based on average plate counts; from the I: 1000 dilutions in the case offield and fruit soils, and from the I :5000 dilution for geocarposphere soil. Weights of soil used in preparation of the dilution series were reasonably constant in 1965, but in the 1966 experiment the weights of soil adhering to the lo-pod samples
Soil mycofiora.D. McDonald
397
varied considerably from week to week and this affected the quantitative results obtained. For geocarposphere soil numbers ofpropagules and mean weights of soil adhering to ten-pod samples are shown in Fig. I. All fungi isolated are listed in Table 2. Results of Warcup plate tests are given for selected fungi in Table 3. The results of the dilution plate tests are presented in Table 4 for field, fruit and geocarposphere soils, the figures shown being percentages of total fungi present at each sampling occasion made up by each species. Numbers of active propagules present in each soil environment are given in Table 5 for a few selected species. 100 ;:;-0 .....
x
~
90
"'"
80
I
VI
ClJ ::l
00
'0c"..
60
00
50
..
c..
c
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'-
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20
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....
I
I
I
,,6
'"
I
I
'd
10 7
8
9
10
11
12
13
14
15
16
Weeks from sowing
Fig.
2.
Numbers offungal propagules per g dry soil. e - e , Field soil; 0- - -0, fruit soil.
From consideration of Table 1 and Fig. 2 it is evident that the fruit soil contained higher numbers of fungal propagules than did the field soil on all but one sampling date. On the single occasion (week 15) when numbers of fungi in field soil were about the same as numbers in fruit soil, it was noted that an unusually high number ofpropagules of Aspergillus flavus was recorded from the field soil. As field soil examined on the following week contained much fewer propagules of A. flavus, it was thought that the sample taken on week 15 was atypical, containing numbers of conidia of A. flavus out of all proportion to the vegetative activity of the fungus in the soil at that time. Total fungi showed a steady increase in numbers in fruit soil from week 10 to week 16, but there was only a slight increase in fungal numbers in field soil during the latter part of the sampling period (Fig. 2). Examination of geocarposphere soil showed that numbers of active fungal propagules per g of dry soil varied inversely with the weight of soil adhering to the ro-pod sample (Fig. I). However, the regression coefficient in the analysis of covariance was not significant and no adjustment
39 8
Transactions British Mycological Society Table
2.
List offungi isolatedfromfield (F),fruit (FR), and geocarposphere (G) soils Warcup plates ~
Absidia sp. Acremonium sp. Alternaria spp. Arthrobotrys sp. Aspergillus caluJidus Link A. carneus (Tiegh.) Bloch A.j/auipes (Bain. & Sart.) Thorn & Church A.flavus Link ex Fr. A. fumigatus Fres. A. glaucus Link A. nidulans (Eidarn) Wint. A. niger v. Tiegh. A. oehraceus Wilh. A. tamani Kita A. terreus Thorn A. ustus (Bain.) Thorn & Church A. versicolor (Vuill.) Tirab. Bahusandhika caligans Batista & Upadh. Botryodiplodia theobromae Pat. Botrytis sp. Cephaksporium acremonium Corda C. roseo-griseum Sabena C. spp. Chaetomium atrobrunneum Ames C. seminudum Ames C. spp. Circinella sp. Cladosporium herbarum (Pers.) Link CWl1IinghaT1ll!lla elegans !.encin. Curvularia lunata (Wakk.) Baed. C. spp. Cylindroearpon sp. Fusarium spp. Glioeladium sp. Gonatobotrys sp. Helminthosponum spp. Hendersonula toruloitka Nattr. Hormodendrum sp. Humicola fuseoatra Traaen H. fusisporus Traaen H. grisea Traaen Macrophomina phaseoli (Maubl.) Ashby Monilia sp. Monotospora sp. Mortierella sp. Mucor spp. Myrotheeium indicum Rarna Rao M. verruearia (Alb. & Schw.) Ditrn. Neocosmospora africana. Arx N. vasinfteta E. F. Sm. Nigrospora sp. Oospora sp. PaeciWmyces fusisporus Saks. P. varioti Bain. P. spp. Pellicularia sp. Penicillium citrinum Thorn P. ehrlichii Kleb. P.frequmtans Westling
F
FR
X
X
x
x
Dilution plates A
F
FR
G X
X X
x
x
X
X
x x
x x x
x x x
x x x
x x
x x
x x x x
x x x
x x
x x x
x x x
x x x x
x
x
x
x
x x x x
X
x x x
x
x x x x
x x x
x x x
x x x
x x x
x x x x x x
x
x
x
x
x x x x
x x
x x x x
x x
x x
x x
x x
x x x
x
X
x x
x x x x
x x x
x
x
x
x
x x
x
x
x x x x x
x x x x
x x x x x
Soil mycofiora. D. McDonald Table
2
399
(cant.) Dilution plates
Warcup plates ~
F
P. funiculosum Thorn P. islandicum Thorn P. variabile Sopp. P. spp. Peyronellaea glomerata (Corda) Goidimich Plwma eupyrena Sacco P. prunicola (Opiz) Wr. & Hochapf P. spp. Pullularia pullulans (de Bary) Berkhout Pyrenochaeta sp. Rhizoctonia solani Kuehn Rhizopus arrhizus Fisch. R. stolonifer (Ehrenb. ex Fr.) Lind. R. spp. Sclerotium roifsii Sacco Spicaria sp. Stachybotrys atra Corda S. spp. Stemphylium sp. Syncephalastrum racemosum (Cohn.) Schroet. S. spp. Tetracoccosporium paxianum Szabo Thielavia basicola Zopf. Torula sp. T riclwikrma oviferum Rifai T. viride Pers. ex S. F. Gray Verticillium spp.
FR
A (
FR
G
x
x
x
x
x
x
x x
x x
x
x
x
x
x
x
x
x
x
x
x
x
F
x x x x x x x x x x x x x x
x x
x
x x
x x
x x x x
x x x x x
x x
x
x x
45
39
27
33
51
Table 3. Warcup plate tests. Numbers ofplates (flO) on which fungi were present Weeks from sowing
,
A
Aspergillus jlavus Botryodiplodia theobromae Fusarium spp. Macrophomina phaseoli Penicillium spp. Rhizoctonia solani Trichoderma viride
Soil F FR F FR F FR F FR F FR F FR F FR
7
8
9
10
II
12
13
14 I
I
I
I
1 10 8
3 3 5 4 2 9 10
2 4 9 8 4 6 10 9
0 6 5 8 4 10 10
3 6 7 9 3 5 9 10
3 8 7 7 2 5 10 10
3 8 6 9 2 9 10 10
3 7 7 10 2 4 10 10
2 9 9 10 2 8 10 9
3
2 3
3
2 3
I
I
3 10 10
15
16
3 7 9 10 3 7 10 10
I
3 4 2
2
3 2
I
5 2
3 2 3
Totals (/100) 0 3 21 59 72 84 24 53 98 98 1 II
27 14
F-field soil; FR-fruit soil.
was made to the results. The high numbers of fungal propagules present in the geocarposphere at weeks 15 and 17 reflected the presence of large numbers of active propagules of Penicillium funiculosum and A. flavus respectively.
Table 4, Dilution plate tests, Fungi expressed as percentages of total isolated Field soil. Weeks from sowing
.
, 8
7 Aspergillus cameus A·fiavipes A·fiavus A·fumigatus A. nidulans A. niger A. ochraceus A. tamarii A. ustus A. versicolor Bahusandhika caligans Botryodiplodia theobromae Cephalosporium spp. Chaetomium spp. Cunninghamella elegans Curvularia spp. Fusarium spp. Hendersonula toruloidea Humicola spp. Macrophomina phaseoli Myrothecium spp. Neocosmospora africana No vasinfecta Paecilomyces spp. Penicillium citrinum P. funiculosum P. spp. Phoma spp. Rhizoctonifl solani Rhizopus spp. Stachybotrys spp. Tetracoccosporium paxianum Thielavia basicola Trichoderma spp.
-
-
-
-
2
I
6 6 13 3
37 8 12
-
9
-
-
10
-
-
3
II
16 -
5 15 12 9 I
12
13
14
15
16
-
-
-
-
-
2
2 6
4 6 16
I
-
10 15 8 4 -
-
8 10 -
-
-
-
-
5 3 3 2 2 3 3 4 - - - - - 2 - -
-
-
-
26
-
15
-
-
-
-
-
-
13
4 I
-
-
-
-
-
I
36 -
-
-
-
-
-
-
-
I
II
I
-
-
53
-
-
-
-
-
-
-
48 -
I
-
I
-
-
-
4
-
I
-
-
-
-
-
2 2
2 -
-
2
-
.
t
-
I I
3 13 17
2
II
-
-
-
-
4
2 2 2
- - I -
- - 2 I 6 3 - - - I - I 63
-
-
2 8
-
10
-
2
34
-
-
I
-
6
-
I
-
-
-
-
7 6 6 7
I
-
2
-
2 28 2 2
-
13
14
15
16
-
-
-
-
-
-
-
-
2 5 8 3
2 3
28
-
2 3
I
-
12
I
I I
II
-
-
10
I I
-
I
I
10 2
3 2 4
7 4 3
4 9 7 2
I
I
I
I
6
3
2
-
-
8 2
-
-
5 3
-
I
-
-
36
35
36
33
-19
-
II
-
14 4
-
I
I
I
22
10
5 2
-
I I I
-
-
2
-
-
-
10 5
4 2 4 4
2
2
22 4
10 2 3
-
-
-
-
I
-
-
I -
I
-
-
-
-
6
-
I
2 3
43
33
-
-
24 4
-
-
-
-
I
I
2 20 9
3 2
-
-
+
-
I
-
-
-
7
I
II
-
-
22
- -
9
I
3
-
t
2
I
16
-
I
-
I
17
18
+ + +
I
I
2
I I
+
I
17 3 I
I
-
17 2 5 2
-
4 25
I
-
5
I
-
-
I
-
I I
-
5
I
2 41
-
I
-
-
2 6
I
I
6
3
-
5
2
-
-
7
-
I
-
-
I
-
-
I
-
I
25
14
-
-
-
+ + 4
+
-
9 II
-
I
6
-
-
4
-
I
-
The percentages given in the above table are to the nearest whole number; figures of less than 0'5 % are shown as
3 20
-
-
23 9 3
-
.... - §' - ...,
3 5 I
-
'+'.
I
+ + + -
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....
6 4
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70
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+ ~ + + 16 34 + - ~ c::l I
-
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31 7 14 4
+ 5 I + I I + I I + I 2 2 + 2 + I + + + -
10
2
+
I
I
-
I
31
II
3
+ 9 5 12 + 12 I I 2 + + + I - + - + - + 3 26 2
-
+ I + + -
+ 3
-
-
I
I
-
3 5
-
-
3
7 3
25 4 7 5
I
-
5 2
+ +
-
II
-
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5
+ -
-
3 24 3
-
I I
-
I
2 30 4
-
2 16 2
3 6 3 3
I
-
-
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2
-
I
2
16
I
-
28 3
-
15
I
-
I
-
14
I
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-
13
I
-
I
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12
I
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-
13 4
4
II
-
5 7
5 5
10
+ +
5 8 -
8
,
A
I
8
4
4 2
-
~
Geocarposphere soil. Weeks from sowing
I
-
2 - -
I
-
I
2
2
-
-
2
8
-
,
10
-
-
-
I
3
- - 2 I 6 5 5 2 2 I - - - 2 - I 3 28 - 2 - - I - - - - - - -
4 13
I
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-
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34 38 2 5 - -
I
-
4
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-
9
I
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8
7
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79
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-
- -
-
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6 2 2
-
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4 3 2 3
I
56 63 -
-
-
8 5
2
-
-
2 3 2 2 8
-
-
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29 2 2 2
7
-
-
II
,
Fruit soil. Weeks from sowing
9 I
+ +
-
I
~
~. Q
Soil mycoflora. D. McDonald
401
The list of species (Table 2) illustrates the influence of the isolation technique and culture medium used on the range offungal species isolated, a wider range of species being isolated from both field and fruit soils by the Warcup soil plate method than by the dilution plate technique. Irrespective of isolation technique, totals of 57, 54 and 52 species were found in field, fruit and geocarposphere soils respectively. Of the fifty-two species from geocarposphere soil, twenty-five had been found in fruit soil in the previous year and of these twenty-four had also been isolated from Table 5. Numbers in thousands of active fungal propagules per g of dry soil in field (F),fruit (FR) and geocarposphere (G) soils Weeks from sowing
,
A
7
Aspergillus spp,
F 4'5 FR 2,8 G 0 Aspergillus F 0'2 jiavus FR 0'7 G 0 Aspergillus F 0'9 fumigatus FR 0'9 G 0 Botryodiplodia F 0 theobromae FR 0 G 0 Fusarium F 4'0 spp, FR 15'6 G 0 Macrophomina F 0 FR 0 phaseoli G 0 Penicillium F 0 spp, FR 3'4 G 0 Rhizocfonia F 0 solani FR 0 G 0
8 12'7 10'7 0 0'2 1'9 0 8'2 0'5 0 0 0 0 6'3 17'9 0 0'4 0 0 0 13'8 0 0'2 0 0
9 3,6 7'3 17'0 0'4 2'0 2'7 0 1,6 7'1 0 0 0 6'4 8'2 22'7 0 0'4 9'4 1'3 6'4 3'7 0 0'4 0
10
II
12
13
14
15
16
17
7'9 3'7 19,8
7'0 5'0 15'0 0 0'4 0'4 1,8 0,6 8'7 0 6'2 0
3'3 2'7 14'3 0 0 2'3 0'4 0'2 5'3 0 3'4 11'2
7'1 4'2 47'1 1'5 0'2 2'1 0'3 0'5 21'9 0'5 2'4 6,6
2'7 11'1 72'4 0'5 7'0 7'5 0'3 1'5 52'0 0 4,8 2,6
14'4 14'5 53'4 0'9 6'8 12,8
0 0 199'9 0 0 10 7'8 0 0 24'2 0 0 3,8
9'1 13'9 12'9 0 5'2 3'5 0,8 2'5 3'1 0 0 0
6'9 14'5 36 '2 0'4 8'4 10'3 0'2 3'1 4'1 0 0'4 0'1
16'9 16'8 44'0 0 4'5 5'5 0 12'3 14'7 0 0 1'1
77'1 19'4 23'9 70 '8 2,6 0'5 0'5 1,8 15'7 0 4,8 1'9 8,6 37'S 47'2 1'4 4'4 6'8
11'4 24'0 56 '2 0'3 17,8 23'2 1'1 0'5 8'5 8'4 6,8 224'4 0 0 0 0 0 0'2
0'9 0'4 0'2 2'9 1'1 13,8 0'2 0'4 0 6,8 4'7 19'3 0'2 4'0 4'2 0 2'2 20'2 0 0'2 0
4'2 3'9 17'5 0 1'4 3'1 11'4 3 1'9 28'0 0 4'8 4'9 2'5 29'S 21'7 0'3 0'5 1'4
18
0 0 68'2 0 0 4 2'2 0 0 16'3 0 0 2'0 0 0 0 0 56 '1 58 '8 0 0 0 0 19'4 10'2 0 0 0 0 16'0 16,6 0 0 0 0 0 0
field soil. Species restricted to anyone specific soil environment were not of common occurrence and were not abundant at any time, Some of the more important and more frequently occurring fungi are considered below. The percentage of aspergilli of total fungi varied through the sampling period in both field and fruit soils, In general, the fungi were more abundant and made up higher percentages of total fungi in field than in fruit soil. In the geocarposphere, Aspergillus spp. were dominant on six occasions and on only one time of sampling (week 15) did they fall as low as third position. On Warcup plates the genus was well represented with A. niger and A. fumigatus the most abundant. The percentage of A. flavus in the mycoflora fluctuated throughout the sampling period in both field and fruit soils, but in both soils the highest figures were recorded during weeks 14-16. In the geocarposphere, A. 26
Myc, 53
402
Transactions British Mycological Society
jlavus showed highest levels, in terms of both numbers of propagules and percentages of total fungi, during weeks 16-18. A. jlavus was not found on Warcup plates from field soil but was present on those from fruit soil sampled at weeks 14-16. A. fumigatus was of frequent occurrence in all soil environments, but was more abundant in field than in fruit soil. In the geocarposphere A.fumigatus composed high percentages oftotal aspergilli during weeks 9- 15. A. nidulans composed higher percentages of total fungi in field than in fruit soil. It was more frequent in Warcup plates from field soil than in those from fruit soil. In geocarposphere soil, A. nidulans had its highest percentages on weeks 16 and 17. A. niger was a common species in all soil environments, being present in higher proportions in field than in fruit soil. It was common at all times on Warcup plates prepared from both field and fruit soils. The fungus was present in geocarposphere soil at all times of sampling. A. ochraceus was not of common occurrence in field or fruit soils but was isolated on weeks 7 and 10 in the former and on week 7 in the latter environment. The fungus was not found on Warcup plates. In geocarposphere soil, A. ochraceus was present on all but one sampling occasion but never composed a high proportion of total fungi. Botryodiplodia theobromae was found on weeks 10 and 13 in field soil and on weeks 10-16 in fruit soil. This fungus was more abundant and composed higher percentages of total fungi of fruit than of field soil. In Warcup plate tests B. theobromae was also isolated more often from fruit than from field soil and showed a tendency to increase in abundance as the fruit developed. In the geocarposphere the fungus was not isolated until week 12, but was found on all subsequent samplings. The various isolates of fusaria obtained in these investigations were not identified to species. The fungi were common in all soil environments. As a percentage of total fungi Fusarium spp. were more important in field soil than in fruit soil, but in terms of numbers of active propagules present the reverse was true. In the geocarposphere the fusaria increased in numbers as the fruit developed, but tended to form a lower proportion of the total mycoflora. Macrophomina phaseoli (syn. Sclerotium bataticola) was uncommon in field soil but was generally abundant in fruit soil, where it composed appreciable percentages of the total fungi, as was shown in both dilution and Warcup plates. M. phaseoli increased in numbers of propagules in the geocarposphere as the fruit matured but there was a tendency for its percentage of total fungi to decrease with time although a slight increase was manifest during the last three times of sampling. Penicillium spp. were present in greater numbers and composed higher percentages of total fungi in fruit than in field soil. In the geocarposphere the proportion of Penicillium spp. in the mycoflora increased slightly as the fruit developed, but numbers of propagules increased to a considerably greater degree. The species most frequently isolated were P. citrinum and P. funiculosum. Rhizoctonia solani was present in all soil environments but not common in any. It occurred rather more often in fruit than in field soil.
Soil mycojWra. D. McDonald
403
Trichoderma viride, despite being isolated from field and fruit soils by both dilution plate and Warcup plate techniques, was of very infrequent occurrence. T. viride was not found in geocarposphere soil in 1966, but the related species T. oviferum was isolated on one occasion. DISCUSSION AND CONCLUSIONS
The 1965 experiment showed that the developing fruit had a strong influence on the mycoflora of the soil surrounding it, numbers of fungal propagules being much higher in fruit than in field soil. The I9~6 investigations indicated an increase in numbers of fungal propagules ill the 16 15
14 l>O
c:
'i
13
0
'" 12
E
...
0
11
I II
~
1lI 1lI
~
9
8 7 Fig. 3. Field soil, selected fungi as percentages of total isolates. Key: see Fig. 5.
geocarposphere as the fruit developed. The quantitites of soil adhering to the ten-pod samples fluctuated considerably from week to week, being influenced by soil moisture content and stage of development of the fruit. It is possible that variation in quantity and nature of exudates from the fruit might influence the adherence of soil, either directly or indirectly by affecting fungal and bacterial growth leading to binding together of soil particles. Numbers of fungal propagules estimated per gram of dry soil varied inversely with the amount of soil adhering to the ten-pod sample and this was almost certainly due to diminution of the geocarposphere effect in soil remote from the fruit surface. The percentages of total fungi in field, fruit and geocarposphere soils composed by four important groups of fungi are shown in Figs. 3,4 and 5 respectively. It is evident that the dominant fungi in all three soil environments were Fusarium spp. and Aspergillus spp., the former being predominant in field and fruit soils and the latter in geocarposphere soil. Penicillium spp. and Macrophomina phaseoli were important in fruit and geocarposphere soils but less significant in field soil. Other fungi which were more abundant in fruit and geocarposphere soils than in field soil include 26-2
404
Transactions British Mycological Society
Botryodiplodia theobromae, Myrothecium verrucaria, Phoma spp. and Rhizoctonia solani. The above results differ from those obtained in Georgia, U.S.A., by Jackson (1968) when he investigated the mycoflora of soil washed from the surfaces of groundnut fruits. He sampled the soil from fruits lifted at intervals over a period of 6 weeks leading up to harvest and found that the 16 15 14
~
c
'i
13
0 on
E 12
...0
on
.,:,t. Q) Q)
~
11 10 9 8 7 Fig. 4. Fruit soil, selected fungi as percentages of total isolates. Key: see Fig. 5.
18
17 16 DO
.=~
15
on
14
~
13
o
E o
11 10
f}:;:J Aspergillus spp.
~PeniCillium spp.
I:: :1 Fusarium spp.
•
D
Other fungi
Macrophomina phaseoli
Fig. 5. Geocarposphere soil, selected fungi as percentages of total isolates.
Soil mycoftora. D. McDonaid dominant fungi were, in order of abundance, Penicillium spp., Aspergillus spp. and Fusarium spp. The reason for the different order is unknown but could be due to different soil conditions during fruit maturation. The balance of the penicillia and aspergilli could be influenced by soil temperature, lower temperatures being likely to favour the former. Many fungi were common to fruit and geocarposphere mycofloras in Nigeria and most of these are included in the list compiled by Jackson (1965) of fungi reported on groundnut pods and kernels. A considerable number of the fungi isolated in Nigeria have also been isolated in Israel by Joffe & Borut (1966), who investigated the mycoflora of groundnut fields in different parts of the country. Two interesting species found in Mokwa soil, but not isolated in Israel, are the toxigenic Penicillium citrinum and Aspergillus ochraceus. Most of the aspergilli and penicillia listed in Table 2 are known to be capable of producing toxic metabolites. It is of interest to note that Fusarium spp. have on many occasions been isolated from rotting pods and seeds, Botryodiplodia theobromae and Macrophomina phaseoli are common causes of concealed damage of seeds, and Rhizoctonia solani is a pathogen of roots, pegs and pods. If soil associated with diseased pods were to be examined instead of soil from around undamaged pods, it is likely that the proportions of the more parasitic species in the mycoflora would be much increased. In conclusion, although the techniques used ostensibly influenced the constitution of the mycofloras of field and fruit soils, quantitative and qualitative influences of the fruit upon the fungi in the soil around it were apparent. Numbers of fungal propagules were consistently higher in fruit soil than in field soil and Botryodiplodia theobromae, Macrophomina phaseoli, Myrothecium verrucaria, Penicillium spp., Phoma spp. and Rhizoctonia solani were all stimulated preferentially by the developing fruit. Geocarposphere soil was similar in species composition to fruit soil, but contained higher numbers of active fungal propagules. The presence of an inverse relation between weights of soil sampled and numbers of fungal propagules present in the geocarposphere gave added evidence for the presence of a strong geocarposphere effect. Thanks are due to the Director, Institute for Agricultural Research, for permission to publish this paper. I wish to express my appreciation to Dr Eva R. Sansome, a.B.E., and to Professor Margaret A. Keay for advice and encouragement during this work. Thanks are also due to the Commonwealth Mycological Institute for identification of a large number of fungal isolates. REFERENCES
DRANSFIELD, M. & McDoNALD, D. (1966). The influence of cropping practice and application offarmyard manure on the soil microflora at Samaru, Northern Nigeria. Niger. agric. ]nl 3, 42-55. GARREN, K. H. (1966). Peanut (groundnut) microfloras and pathogenesis in peanut pod rot. Phytopath. Z. 55, 359-367. HILTNER, L. (1904). Uber neuere Erfahrungen und Probleme auf dem Gebiet der Bodenbakteriologie und unter besonderer Beriicksichtigung der Griindiingung und Brache. Arb. dt. LandwGes. ga, 59-78.
Transactions British Mycological Society C. R. (1965). A list of fungi reported on peanut pods and kernels. Mimeogr. Ser Ga Coastal Plain Exp. Stn N.S. 234. JACKSON, C. R. (1968). A field study of fungal associations on peanut fruit. Res. Bull. Univ. Ga agric. Exp. Stn no. 26. JOFFE, A. Z. & BoRUT, SmRAY (1966). Soil and kernel mycoflora of groundnut B.elds in Israel. Mycologia 58, 62g-640. RAo, A. S. (1962). Fungal populations in the rhizosphere of peanut (Arachis hypogaea L.). Pl. Soil.7, 2, 260-266. WARCUP, J. H. (1950). The soil-plate method for isolation of fungi from soil. Nature, Lond. • 66, II? WILLIAMS, L. E. & SCHMITTENHENNER, A. F. (1960). Effect of growing crops and crop residues on soil fungi and seedling blights. Phytopathology 50, 22-25. JACKSON,
(Accepted for publication
22
June 1969)