- Email: [email protected]
Seed mycofloras of hyacinth beans (Lablab niger) and long beans (Vigna sesquipedalis) and their pathogenic importance
[ 5°3 ] Trans. Br. mycol. Soc. 78 (3) 503-508 (1982)
Printed in Great Britain
SEED MYCOFLORAS OF HYACINTH BEANS (LABLAB NIGER) AND LONG BEANS (VIGNA SESQUIPEDALIS) AND THEIR PATHOGENIC IMPORTANCE By VIJAYA S. KANAPATHIPILLAI Department of Biology, Agricultural University of Malaysia, Serdang, Selangor, Malaysia
Isolations and identifications of seed-borne fungi from two seed lots of Lablab niger and Vigna sesquipedalis seed were made. Among the various species of fungi isolated, Curvularia sp., Drechslera sativum, Diplodia sp., Chaetomium globosum, Pestalotiopsis sydowiana, Aspergillus spp. and Fusarium spp. were the most common on these two beans. Nigrospora sacchari and Phyllosticta sp. were confined to L. niger. Macrophomina phaseolina and Colletotrichum sp. were found only on V. sesquipedalis. Selected fungi were isolated, and tested for pathogenicity to emerging seedlings and established seedlings of L. niger and V. sesquipedalis. Phyllostieta sp., Diplodia sp., Drechslera sativum and Macrophomina phaseolina were found to be pathogenic. The pathological importance of these fungi is further discussed. Lablab niger Medik, commonly called hyacinth bean or Egyptian bean and asparagus pea or yard-long bean, Vigna sesquipedalis (L.) Fraus, are two widely cultivated crops in the Far East and Africa. The International Seed Testing Association (I.S.T.A.), Annotated List of Seed-Borne Diseases (Noble & Richardson, 1968) records Colletotrichum lindemuthianum (Sacc, & Magn.) Br. & Cav. as a seed-borne pathogen of L. niger and Ascochyta phaseolorum Sacc., Diaporthe phaseolorum (Cooke & Ellis) Sacco var. sojae (Lehm.) Wehm., Macrophomina phaseolina (Tassi) Goid., Cladosporium vignae Gardner, Corynespora cassiicola (Berk. & Curt.) Wei and Fusarium oxysporum Schlecht. ex Fr. f.sp. tracheiphilum (E. F. Sm.) Snyd. & Hans. as seedborne pathogens of V. sesquipedalis. Seed testing for purity, germination rate and seed health has become an accepted practice in agriculture and horticulture in many countries. In Malaysia, very little work has been done on this aspect and there have been few attempts to produce quality seeds in bulk. Because little is known of the seed-borne fungi of these two plants a study was made of selected seed samples from crops grown in Malaysia by isolating the fungi and testing their pathogenicity. MATERIALS AND METHODS
I solation studies
Two lots of seed samples of V. sesquipedalis and L. niger were obtained from the Faculty of Agriculture, University of Malaya, on two occasions. The seeds were placed on 2 % malt extract agar (MA) before and after pretreatment with 10% Chlorox (1 %
available chlorine). A total of 100 seeds, selected at random from each sample were used with two seeds per Petri dish and incubated at 25°C for one week. The dishes were examined after 7 days.
Pathogenicity tests
Fungi isolated from the seeds were tested for their pathogenicity to emerging seedlings and established seedlings of the two leguminous hosts in a glasshouse. Emerging seedlings: soil inoculation. Plastic pots (5 in diam) containing John Innes Compost No.2 were inoculated with fungi isolated previously from the seeds (Table 1), using one dish of culture per pot as the inoculum. The pots containing the infested compost were covered with polythene sheets for 2 days before five seeds/pot were sown c. 2'5 em below the compost surface. There were two replicates. The pots were again covered for 2 days with polythene sheets. Seedlings emerged 5-7 days after sowing, and 10 days after sowing were examined for disease symptoms. Seedling infection at the trifoliate stage. Two Petri dish cultures of fungi, selected from the previous test, were inoculated on seedlings at the trifoliate stage. Two dishes of each fungal culture were homogenized in 200 ml of sterile distilled water. This suspension was poured over the entire plant surface of each host, ensuring that the inoculum covered surfaces of leaf, stem and apical region. Each treatment consisted of two seedlings per pot, the total of four seedlings. The test was repeated. Disease symptoms were observed after 14-28 days. Assessment of the damage done and description of
Mycofl ora of hyacinth and long beans
5°4
Table
1.
Fungi isolated f rom seeds of Labl ab niger
Frequency of occurrence U u Sample 2
Sample 1 Fungi isolated Aspergillus chevalieri A . fiavus A. j anus A . japonicus A . ochraceus A. niger A. uientii A. tamarii Chaetomium globosum Choanephora cucurbitarum Cladosporium cladosporioides Curvula ria eragrostidis C. inaequalis C. lunata var. aeria C. senagalensis Diplodia sp. Drechslera sativa Diaporthe phaseolorum Fusarium equiseti F. sambucinum M ucor sp. N igrospora sacchari Pestalotiopsis sydounana
Penicillium wortmanii Pseudobotry tis sp. Phyllosticta sp . Synceph alastrum sp.
Untreat ed
Chloros pretreatment
Untreated
Chloros pretreatment
7'5 5'0
4 '0
5'0 7' 5
2'0
0 ,8
1'7 8'3 3'3 8'3
8'3 2'0
5'0
2 '0
29' 2 5' 8
2 '0
7'5
4 '0
5 '0
6·6
0 ·8 0,8
0,8
2 '0
5'0 0 ,8 15 '0
8'3 15'0
2 '0 8 '0 4 '0
30 ' 0
12'5 7'5 21'7 2'5 1'7 2'5 1'7 1'7
8 '0
0· 8 20 '0 4' 0
2 '0 2 '0
0 ·8
52 '0
4 8'0
6 '0
0 ,8
Trichoderma viride
Germination
10 '0
20 '0
sympto m s were made for each fungus inoculate d. As a control treatment, one set of plants were sprayed with di stilled water and another with homogenized malt -agar suspension.
10 0'0
only and C haetomium sp ., D iap orth e sp ., Nigros pora sp., Ph oma sp ., P seudobotry tis sp . and Syncep halastr um sp. were found onl y on L. niger . Pathogen ic impo rtance of the fungi isolated from the seed samp les is sho wn in Tables 3 and 4. Results indicate that most isolates tested were RESULTS pathogenic to the host plants especially to L . niger. The fungi isolated from L. niger and V . sesquip edal is In all cases, cont rol plants appeared healthy and normal. are given in Tables 1 and 2 . The most commo n species of fungi were Chaetomiumglobosum . The germination of seed of A sp ergillus, C hoanep ho ra, C urvularia, Drechslera , both host pl ants was not grea tly hampered by thi s D iplodia, Fusarium and Mucor . Other speci es of fungus and seedlings th at emerged did not sho w fungi occasio na lly isolated belonged to the genera any visible symptoms of disease. This fungus was Co lleto trichum, C haeto miu m , D iap orth e, Mac ropho- not tested further for its pathogenicit y. Colletotrichum lindemuth ianum. This was not mina , Nig rospora , Pestalotiopsis, Phoma , Phyllosticta, Pseudobotry tis and Syncephalastrum , The used in soil expe rime nts bu t was sp rayed on larger genera commo n to both host s were : A spergill us, seedlings of L. niger, whic h show ed 20 % leaf-area C hoa nep hora, Curvul aria, Diplodia, Drechslera, F us- severity of leaf symptom s on 86 % of the leaflets. arium , Mucor and Pestalotiopsis. Macrophomina sp . Both mon ofoliate and trifoliate leave s showed and C olletotrichu m sp. were present in Vigna seeds severe leaf symptom s. The lesions were br own in
Vijaya S. Kanapathipillai Table
2.
5°5
Fungi isolated from seeds of Vigna sesquipedalis Frequency of occurrence % Sample
Fungi isolated
Untreated 0·8 1"7 0·8 0·8 0"8
Aspergillus f/avus
A. fumigatus A.janus A. ochraceus A. niveus A. niger A. versicolor A. wentii Choanephora cucurbitarum Chaetomium globosum Colletotrichum lindemuthianum Curvularia lunata var. aeria C. senegalensis Diplodia sp. Drechslera sativum Fusarium moniliforme Mucor sp. Macrophomina phaseolina Pestalotiopsis sydowiana Penicillium wortmanii Syncephalastrum sp. Trichoderma viride
Sample
1
Chloros pretreatment
Untreated
2
Chloros pretreatment
2
4'1 1"7 4"1
6'0
29"2
4'0
8"3 1'7 1'7 2"5
5'0
6·6
10·8
2'5 5"8 2"5
11"7
16'0
3'3
2'0 16'0
4"0
0·8 2
12'5
6"6
4'0 5"0
4"1
0"8
Germination
95
69
100
90
Table 3. Results of pathogenicity test on emergent seedlings of Lablab niger and Vigna sesquipedalis Lablab niger*
Fungi isolated None - Control Chaetomium globosum Curvularia inaequalis C. lunata var. aeria C. senegalensis
Seedling emergence (maximum 10) 10
8 9 10 10
Diplodia sp.
9
Drechslera sativum Fusarium equiseti
9 9
F. moniliforme
4
F. sambucinum
8 9
Macrophomina phaseolina Nigrospora sacchari Trichoderma viride
10
8
*
Pathogenic symptoms None None None, slow germination Brown leaf spots (2 plants) None
Vigna sesquipedalis*
Seedling emergence (maximum 10)
Pre-emergent death (4 seedlings) Distortion of leaf centres Seedlings wilting brown leaf spots One plant wilting others brown leaf spots None Brown leaf spots None None
Observed on 5 seedlings/pot/z replicate.
9 9 8 9 10 10
8 9 10 10
8 9 8
Pathogenic symptoms None None None Brown leaf spots Brown leaf spots with scorched edges None Brown leaf spots Brown leaf spots wrinkled leaf surface Brown leaf spots Brown leaf spots Brown leaf spots None None
506
Mycofiora of hyacinth and long beans Table 4. Pathogenicity of selected isolates on seedlings at trifoliate stage Infection" and severity] Lablab niger
Fungi tested Colletotrichum lindemutianum Curvularia inaequalis C. lunata var. aeria
C. senegalensis Diplodia sp . Drechslera sativum Fusarium equiseti F . moniliforme F. sambucinum Macrophomina phaseolina Pestalotiopsis sydowiana Phyllostieta sp,
*
Vigna sesq uipedalis
Infection
Severity
Infection
Severity
(% )
(%)
('Yo)
('Yo)
86 72
++
60
+
67 17
66 100
80 91
65 85 65 95
+
+ + + ++++ ++ + ++
(one dead plant)
+
+++
° °
°
° °
+
12
(three dead plants)
+ +
4
3°
number of leaflets affected x 100. total number of leaflets t Severity = visual estimation of the area of the affected leaflet : -, no leaf symptoms; 20 % leaflet area; + + +, 30 % leaflet area; + + + +, more than 30 % leaflet area . '_<"
% inrecnon
+ + +
37 6 5°
=
colour and more frequent along the veins of the lower surface than on the upper surface. Severely infected leaves wrinkled and dried. Under microscopic examination of sections of diseased portions, light-coloured sclerotia were seen within the epidermal and some palisade cells. Similarly, on V. sesquipedalis, there was veinal necrosis as well as tiny, crowded reddish brown spots on the leaf surface. Curuularia inaequalis . This had very little effect on seed germination of both host plants except that germination of L. niger seeds was much slower than in the control experiments. Seedlings appeared normal, without any external disease symptoms. In the second seedling infection experiment with the fungus, small dark-brown spots occurred on most of the trifoliate and monofoliate leaves but the spots were discrete and did not damage the leaves. Whereas on V. sesquipedalis seedlings there was no visible symptom. Curvularia lunata var. aeria had no effect on the germination of seeds, though a few brown leaf spots were seen on the leaves of the seedlings of both hosts. In seedling-infection studies few discrete dark spots were seen on trifoliate leaves of L. niger only. Curuularia senegalensis had little effect on seed germination of both hosts and few brown spots were only seen on the leaves of V. sesquipedalis, though no symptoms were seen with seedling inoculations. Only a small percentage ofthe leaflets of L. niger had some spots.
+,
10 %
leaflet area;
+ +,
Of nine seeds of L. niger germinated on soil infected with Diplodia sp., four died just before emergence while the other seedlings had brown leaf spots . After seedling inoculations, a tenth of the leaflet area infection on about two-thirds of the leaflets were seen as brownish black spots with chlorotic rims, the centres of which were dry. However, seed germination of V . sesquipedalis was not affected and no leaf symptoms were seen on seedling inoculations. The pre-emergent death of seedlings indicate that this isolate, which is placed as Diplodia sp., is pathogenic to Lablab niger. The fungus forms a few black, cylindrical, pycnidia 1 em high and 4 mm diam in culture. Conidiophores are short, hyaline 35-70 pm high and 31-5'6 pm wide ; conidia are large, ellipsoidal to subcylindrical, measuring 25'5 (35'5) 45'5 x 14'6 (16'4) 20'0 pm, hyaline becoming brown black and thick-walled, non-septate to i-septate. Brown leaf spots were present and leaf centres were distorted for seedlings of L. niger emerging from Drechslera sativa infested soil. This fungus caused severe leaf symptoms on this host plant; monofoliate leaves dying off and the trifoliate leaves being covered with large irregularly shaped spots, with desiccated centres, and wrinkled leaf surfaces. Relatively high susceptibility to infection with this fungus was seen on seedlings of V. sesquipedalis. The germination of both host seeds was not impaired by this fungus.
Vijaya S. Kanapathipillai
5°7
The seedlings of L. niger emerging from soil Seedlings inoculated with P. sydowiana showed infested with Fusarium equiseti wilted after 10 days very few leaf spots which were black coloured, on and those of V . sesquipedalis showed brown leaf both hosts; the younger leaves were more spots and wrinkled leaf surfaces. Seedling inocula- susceptible. tion of V. sesquipedalis showed no visible symptoms. Severe infection was seen on the leaves of L. niger DISCUSSION seedlings after seedling inoculation, with leaves being badly damaged by large, irregular brown The seeds of L. niger were infested with about 27 lesions with dry centres. fungi and those of V. sesquipedalis with about 22 Though few brown spots were present on some fungi. Among these, Colletotr ichum lindemuthianum, leaves of seedlings of V . sesquipedalis emerging from Ma crophomina phaseolina and Diaporthe phaseosoil infested with F. moniliforme, no other symptoms lorum are the only fungi reported by Noble & were seen on larger seedlings after spraying them Richardson (1968) to be seed-borne pathogens. with the fungus. However, four out of ten seeds of Species of Aspergillus, Penicillium, Mucor, ChoaneL. niger germinated, one of which later wilted. On phora , Syncephalastrum and a few others can be larger seedlings, only minute black spots were seen considered as storage fungi or non-pathogenic on leaves. The fungus infection occurs mostly at fungi, commonly found on the seeds . Other fungi germination but not on larger plants. The fungus recorded here are seed-borne fungi which may be reduces germination, pre-emergence and probably pathogenic to the host plants. Colletotrichum sp., isolated from seeds of V. causes wilting. The leaf spotting in these experiments is of little importance as compared with the sesquipedalis, caused veinal necrosis of both host plants, L. niger being more susceptible. It is reduction in germination and emergence. Fusarium sambucinum was seen to have little recorded as a seed-borne pathogen of L. niger effect on germination of seeds of both hosts. After (N oble & Richardson, 1968) and is parasitic on seedling inoculation with this fungus, only a few other leguminous plants including Vigna sp. leaf spots were seen on V . sesquipedalis but very (C. M .I. distribution Map. No. 177; Sristava & large light-brown lesions with thin but distinct dark Mathur, 1954). It causes anthracnose of pods and brown borders were seen on the leaf margins of seeds of leguminous crops. L. niger. Ma crophomina phaseolina was isolated from seeds Brown leaf spots were seen on both host of V . sesquipedalis and was found to be pathogenic seedlings emerging from soil infested with Macro- to both host seedlings, wounded and unwounded. phomina phaseolina. In seedling infection trial, It is reported on many different host plants, L. niger showed severe leaf spots, portions of stems including Vigna sp., L. niger, is also found in soil and petioles started to rot , and one plant died after and is a seed-borne pathogen of V . sinensis (L utt relJ 8 weeks. Black sclerotia and pycnidia were found & Weimer, 1952; Sackston, 1969 ; Tehon & Bowe, on the surface of stems of the dead portions. One 1939; Wallace, 1932). This fungus causes charcoal other plant ceased to grow ; young leaves were rot, ashy stem blight or stem canker of seedling etiolated and stems rotted, On V . sesquipedalis, no beans and is injurious to seed lings devitalized by leaf spots were seen, but three plants shed leaves other factors (H offmaster, Maclaughlin, Ray & and died 2 months after inoculation. Areas of stem Chester, 1943). Pycnidial formation on diseased were brown in colour and black sclerotia and portions found here, has also been reported by pycnidia formed on the infected surface. Labora- others (Bhargva, 1965; Ghaffar & Kafi, 1965; tory experiments using this fungus on both host Mathur, 1967). seedlings, wounded and non-wounded, showed Diaporthe phaseolorum was isolated from seeds of that this fungus infects some unwounded seedlings L. niger and was previously reported to be a seed-borne pathogen of V. sesquipedalis. As infection but wounding led to almost 100 % infection. As the seedlings emerging from soil infested with studies were not carried out with this fungus, the Nigrospora sacchari were normal and healthy, this pathogenicity of this fungus is not reported. Other fungi parasitizing seeds and seedlings of fungus was not tested in seedling infection studies. Whereas, Phyllostieta sp ., which was not tested in the host plants are Diplodia sp ., Drechslera sativum, soil inoculation studies, promoted severe leaf- Fusarium equiseti, F. moniliforme and Phyllosticta infection symptoms on seedlings of L. niger and sp. Some seedlings emerging from soil infested some seedlings of V. sesquipedalis. Heavily infested with Diplodia sp. were killed, otherwise few leaves were dead and others withered. The spots leafspot symptoms were seen on seedling inocuwere dark brown in colour with chlorotic margins lation ; also wounding encourages infection of the enlarging to about 10-15 mm. Species identification host plants by this fungus. Dr echslera sativum and Phyllosticta sp . caused more severe leaf spot on of this isolate was not made.
508
Mycoflora of hyacinth and long beans
seedlings of L. niger than on V. sesquipedalis. Seedlings of L. niger emerging from soil inoculated with F. equiseti, died and the leaves of seedlings inoculated with the fungi showed leaf spots. F. monilijorme reduced germination of seeds and caused wilting of one of the emerged seedling. The following fungi had very little or no effect on the infection of seeds or seedlings: Curvularia inaequalis, C. lunata, C. senegalensis, Fusarium sambucinum, Chaetomium globosum and Nigrospora sacchari. Most of the fungi tested for pathogenicity, sporulate well in culture except Macrophomina phaseolina. Hence use of whole culture suspension, i.e. fungi plus added nutrients from media, was found to be a more convenient and uniform method. The control plants sprayed with water or sprayed with agar suspension, were normal and healthy. The chloros pretreatment did reduce the incidence of a number of fungi, which may be superficially present on the seeds.
REFERENCES
BHARGVA, S. N. (1965). Studies on charcoal rot of Potatoes. Phytopathologische Zeitschrift 53, 35-54· C.M.!. DISTRIBUTION MAP No. 177. . GHAFFAR, A. & KAFt, A. (1965). Dolichos lablab, a new
pycnidial host of Macrophomina phaseoli (Maubl.) Ashby. Current Science 34, 515. HOFFMASTER, D. E., MACLAUGHLIN, J. M., RAy, W. W. & CHESTER, K. S. (1943). The problem of dry rot caused by Macrophomina phaseoli (Sclerotium bataticoiai. Phytopathology 33, 1113-1114 (Abstr.). KANAPATHIPILLAI, V. S. (1972). The effect of systemic fungicides on U. nuda in barley and P. avenae in oats, together with a study of the fungal flora of seeds of some Malaysian crops. Ph.D. Thesis, Queen's University, Belfast. LUTTRELL, E. S. & WEIMER, J. L. (1952). Macrophomina phaseoli: a stem canker and ashy stem blight of cowpea. Plant Disease Reporter 36, 194-195. MATHUR, S. B. (1967). Sclerotia and pycnidia of Macrophomina phaseoli developed on stems of cowpea, beans (Phaseolusvulgaris) and cotton. Phytopathologische Zeitschrift 58, 137-140. NOBLE, M. & RtCHARDSON, M. J. (1968). An annotated list of seed-borne diseases. Proceedings of International Seed Testing Association 33, 1-191. SACKSTON, W. E. (1969). Sclerotia bataticola on seeds of cowpea, Vigna sinensis. Plant Disease Reporter 53, 438-439. SRISTAVA, H. C. & MATHUR, P. B. (1954). Cold storage of field beans (Dolichos lablab). Indian Journal of Horticulture 11, 125-128. TEHON, L. R. & HOWE, G. H. (1939). Charcoal rot in Illinois. Plant Disease Reporter z3, 312-325. WALLACE, G. B. (1932). Report of the Mycologist. Ann. Rep. Dept. Agric. Tanganyika Territory, 1931 PP· 94-97. (Abstract Review of Applied Mycology u, 201).
(Reviewed for publication 14 May 1981)
Printed in Great Britain
SEED MYCOFLORAS OF HYACINTH BEANS (LABLAB NIGER) AND LONG BEANS (VIGNA SESQUIPEDALIS) AND THEIR PATHOGENIC IMPORTANCE By VIJAYA S. KANAPATHIPILLAI Department of Biology, Agricultural University of Malaysia, Serdang, Selangor, Malaysia
Isolations and identifications of seed-borne fungi from two seed lots of Lablab niger and Vigna sesquipedalis seed were made. Among the various species of fungi isolated, Curvularia sp., Drechslera sativum, Diplodia sp., Chaetomium globosum, Pestalotiopsis sydowiana, Aspergillus spp. and Fusarium spp. were the most common on these two beans. Nigrospora sacchari and Phyllosticta sp. were confined to L. niger. Macrophomina phaseolina and Colletotrichum sp. were found only on V. sesquipedalis. Selected fungi were isolated, and tested for pathogenicity to emerging seedlings and established seedlings of L. niger and V. sesquipedalis. Phyllostieta sp., Diplodia sp., Drechslera sativum and Macrophomina phaseolina were found to be pathogenic. The pathological importance of these fungi is further discussed. Lablab niger Medik, commonly called hyacinth bean or Egyptian bean and asparagus pea or yard-long bean, Vigna sesquipedalis (L.) Fraus, are two widely cultivated crops in the Far East and Africa. The International Seed Testing Association (I.S.T.A.), Annotated List of Seed-Borne Diseases (Noble & Richardson, 1968) records Colletotrichum lindemuthianum (Sacc, & Magn.) Br. & Cav. as a seed-borne pathogen of L. niger and Ascochyta phaseolorum Sacc., Diaporthe phaseolorum (Cooke & Ellis) Sacco var. sojae (Lehm.) Wehm., Macrophomina phaseolina (Tassi) Goid., Cladosporium vignae Gardner, Corynespora cassiicola (Berk. & Curt.) Wei and Fusarium oxysporum Schlecht. ex Fr. f.sp. tracheiphilum (E. F. Sm.) Snyd. & Hans. as seedborne pathogens of V. sesquipedalis. Seed testing for purity, germination rate and seed health has become an accepted practice in agriculture and horticulture in many countries. In Malaysia, very little work has been done on this aspect and there have been few attempts to produce quality seeds in bulk. Because little is known of the seed-borne fungi of these two plants a study was made of selected seed samples from crops grown in Malaysia by isolating the fungi and testing their pathogenicity. MATERIALS AND METHODS
I solation studies
Two lots of seed samples of V. sesquipedalis and L. niger were obtained from the Faculty of Agriculture, University of Malaya, on two occasions. The seeds were placed on 2 % malt extract agar (MA) before and after pretreatment with 10% Chlorox (1 %
available chlorine). A total of 100 seeds, selected at random from each sample were used with two seeds per Petri dish and incubated at 25°C for one week. The dishes were examined after 7 days.
Pathogenicity tests
Fungi isolated from the seeds were tested for their pathogenicity to emerging seedlings and established seedlings of the two leguminous hosts in a glasshouse. Emerging seedlings: soil inoculation. Plastic pots (5 in diam) containing John Innes Compost No.2 were inoculated with fungi isolated previously from the seeds (Table 1), using one dish of culture per pot as the inoculum. The pots containing the infested compost were covered with polythene sheets for 2 days before five seeds/pot were sown c. 2'5 em below the compost surface. There were two replicates. The pots were again covered for 2 days with polythene sheets. Seedlings emerged 5-7 days after sowing, and 10 days after sowing were examined for disease symptoms. Seedling infection at the trifoliate stage. Two Petri dish cultures of fungi, selected from the previous test, were inoculated on seedlings at the trifoliate stage. Two dishes of each fungal culture were homogenized in 200 ml of sterile distilled water. This suspension was poured over the entire plant surface of each host, ensuring that the inoculum covered surfaces of leaf, stem and apical region. Each treatment consisted of two seedlings per pot, the total of four seedlings. The test was repeated. Disease symptoms were observed after 14-28 days. Assessment of the damage done and description of
Mycofl ora of hyacinth and long beans
5°4
Table
1.
Fungi isolated f rom seeds of Labl ab niger
Frequency of occurrence U u Sample 2
Sample 1 Fungi isolated Aspergillus chevalieri A . fiavus A. j anus A . japonicus A . ochraceus A. niger A. uientii A. tamarii Chaetomium globosum Choanephora cucurbitarum Cladosporium cladosporioides Curvula ria eragrostidis C. inaequalis C. lunata var. aeria C. senagalensis Diplodia sp. Drechslera sativa Diaporthe phaseolorum Fusarium equiseti F. sambucinum M ucor sp. N igrospora sacchari Pestalotiopsis sydounana
Penicillium wortmanii Pseudobotry tis sp. Phyllosticta sp . Synceph alastrum sp.
Untreat ed
Chloros pretreatment
Untreated
Chloros pretreatment
7'5 5'0
4 '0
5'0 7' 5
2'0
0 ,8
1'7 8'3 3'3 8'3
8'3 2'0
5'0
2 '0
29' 2 5' 8
2 '0
7'5
4 '0
5 '0
6·6
0 ·8 0,8
0,8
2 '0
5'0 0 ,8 15 '0
8'3 15'0
2 '0 8 '0 4 '0
30 ' 0
12'5 7'5 21'7 2'5 1'7 2'5 1'7 1'7
8 '0
0· 8 20 '0 4' 0
2 '0 2 '0
0 ·8
52 '0
4 8'0
6 '0
0 ,8
Trichoderma viride
Germination
10 '0
20 '0
sympto m s were made for each fungus inoculate d. As a control treatment, one set of plants were sprayed with di stilled water and another with homogenized malt -agar suspension.
10 0'0
only and C haetomium sp ., D iap orth e sp ., Nigros pora sp., Ph oma sp ., P seudobotry tis sp . and Syncep halastr um sp. were found onl y on L. niger . Pathogen ic impo rtance of the fungi isolated from the seed samp les is sho wn in Tables 3 and 4. Results indicate that most isolates tested were RESULTS pathogenic to the host plants especially to L . niger. The fungi isolated from L. niger and V . sesquip edal is In all cases, cont rol plants appeared healthy and normal. are given in Tables 1 and 2 . The most commo n species of fungi were Chaetomiumglobosum . The germination of seed of A sp ergillus, C hoanep ho ra, C urvularia, Drechslera , both host pl ants was not grea tly hampered by thi s D iplodia, Fusarium and Mucor . Other speci es of fungus and seedlings th at emerged did not sho w fungi occasio na lly isolated belonged to the genera any visible symptoms of disease. This fungus was Co lleto trichum, C haeto miu m , D iap orth e, Mac ropho- not tested further for its pathogenicit y. Colletotrichum lindemuth ianum. This was not mina , Nig rospora , Pestalotiopsis, Phoma , Phyllosticta, Pseudobotry tis and Syncephalastrum , The used in soil expe rime nts bu t was sp rayed on larger genera commo n to both host s were : A spergill us, seedlings of L. niger, whic h show ed 20 % leaf-area C hoa nep hora, Curvul aria, Diplodia, Drechslera, F us- severity of leaf symptom s on 86 % of the leaflets. arium , Mucor and Pestalotiopsis. Macrophomina sp . Both mon ofoliate and trifoliate leave s showed and C olletotrichu m sp. were present in Vigna seeds severe leaf symptom s. The lesions were br own in
Vijaya S. Kanapathipillai Table
2.
5°5
Fungi isolated from seeds of Vigna sesquipedalis Frequency of occurrence % Sample
Fungi isolated
Untreated 0·8 1"7 0·8 0·8 0"8
Aspergillus f/avus
A. fumigatus A.janus A. ochraceus A. niveus A. niger A. versicolor A. wentii Choanephora cucurbitarum Chaetomium globosum Colletotrichum lindemuthianum Curvularia lunata var. aeria C. senegalensis Diplodia sp. Drechslera sativum Fusarium moniliforme Mucor sp. Macrophomina phaseolina Pestalotiopsis sydowiana Penicillium wortmanii Syncephalastrum sp. Trichoderma viride
Sample
1
Chloros pretreatment
Untreated
2
Chloros pretreatment
2
4'1 1"7 4"1
6'0
29"2
4'0
8"3 1'7 1'7 2"5
5'0
6·6
10·8
2'5 5"8 2"5
11"7
16'0
3'3
2'0 16'0
4"0
0·8 2
12'5
6"6
4'0 5"0
4"1
0"8
Germination
95
69
100
90
Table 3. Results of pathogenicity test on emergent seedlings of Lablab niger and Vigna sesquipedalis Lablab niger*
Fungi isolated None - Control Chaetomium globosum Curvularia inaequalis C. lunata var. aeria C. senegalensis
Seedling emergence (maximum 10) 10
8 9 10 10
Diplodia sp.
9
Drechslera sativum Fusarium equiseti
9 9
F. moniliforme
4
F. sambucinum
8 9
Macrophomina phaseolina Nigrospora sacchari Trichoderma viride
10
8
*
Pathogenic symptoms None None None, slow germination Brown leaf spots (2 plants) None
Vigna sesquipedalis*
Seedling emergence (maximum 10)
Pre-emergent death (4 seedlings) Distortion of leaf centres Seedlings wilting brown leaf spots One plant wilting others brown leaf spots None Brown leaf spots None None
Observed on 5 seedlings/pot/z replicate.
9 9 8 9 10 10
8 9 10 10
8 9 8
Pathogenic symptoms None None None Brown leaf spots Brown leaf spots with scorched edges None Brown leaf spots Brown leaf spots wrinkled leaf surface Brown leaf spots Brown leaf spots Brown leaf spots None None
506
Mycofiora of hyacinth and long beans Table 4. Pathogenicity of selected isolates on seedlings at trifoliate stage Infection" and severity] Lablab niger
Fungi tested Colletotrichum lindemutianum Curvularia inaequalis C. lunata var. aeria
C. senegalensis Diplodia sp . Drechslera sativum Fusarium equiseti F . moniliforme F. sambucinum Macrophomina phaseolina Pestalotiopsis sydowiana Phyllostieta sp,
*
Vigna sesq uipedalis
Infection
Severity
Infection
Severity
(% )
(%)
('Yo)
('Yo)
86 72
++
60
+
67 17
66 100
80 91
65 85 65 95
+
+ + + ++++ ++ + ++
(one dead plant)
+
+++
° °
°
° °
+
12
(three dead plants)
+ +
4
3°
number of leaflets affected x 100. total number of leaflets t Severity = visual estimation of the area of the affected leaflet : -, no leaf symptoms; 20 % leaflet area; + + +, 30 % leaflet area; + + + +, more than 30 % leaflet area . '_<"
% inrecnon
+ + +
37 6 5°
=
colour and more frequent along the veins of the lower surface than on the upper surface. Severely infected leaves wrinkled and dried. Under microscopic examination of sections of diseased portions, light-coloured sclerotia were seen within the epidermal and some palisade cells. Similarly, on V. sesquipedalis, there was veinal necrosis as well as tiny, crowded reddish brown spots on the leaf surface. Curuularia inaequalis . This had very little effect on seed germination of both host plants except that germination of L. niger seeds was much slower than in the control experiments. Seedlings appeared normal, without any external disease symptoms. In the second seedling infection experiment with the fungus, small dark-brown spots occurred on most of the trifoliate and monofoliate leaves but the spots were discrete and did not damage the leaves. Whereas on V. sesquipedalis seedlings there was no visible symptom. Curvularia lunata var. aeria had no effect on the germination of seeds, though a few brown leaf spots were seen on the leaves of the seedlings of both hosts. In seedling-infection studies few discrete dark spots were seen on trifoliate leaves of L. niger only. Curuularia senegalensis had little effect on seed germination of both hosts and few brown spots were only seen on the leaves of V. sesquipedalis, though no symptoms were seen with seedling inoculations. Only a small percentage ofthe leaflets of L. niger had some spots.
+,
10 %
leaflet area;
+ +,
Of nine seeds of L. niger germinated on soil infected with Diplodia sp., four died just before emergence while the other seedlings had brown leaf spots . After seedling inoculations, a tenth of the leaflet area infection on about two-thirds of the leaflets were seen as brownish black spots with chlorotic rims, the centres of which were dry. However, seed germination of V . sesquipedalis was not affected and no leaf symptoms were seen on seedling inoculations. The pre-emergent death of seedlings indicate that this isolate, which is placed as Diplodia sp., is pathogenic to Lablab niger. The fungus forms a few black, cylindrical, pycnidia 1 em high and 4 mm diam in culture. Conidiophores are short, hyaline 35-70 pm high and 31-5'6 pm wide ; conidia are large, ellipsoidal to subcylindrical, measuring 25'5 (35'5) 45'5 x 14'6 (16'4) 20'0 pm, hyaline becoming brown black and thick-walled, non-septate to i-septate. Brown leaf spots were present and leaf centres were distorted for seedlings of L. niger emerging from Drechslera sativa infested soil. This fungus caused severe leaf symptoms on this host plant; monofoliate leaves dying off and the trifoliate leaves being covered with large irregularly shaped spots, with desiccated centres, and wrinkled leaf surfaces. Relatively high susceptibility to infection with this fungus was seen on seedlings of V. sesquipedalis. The germination of both host seeds was not impaired by this fungus.
Vijaya S. Kanapathipillai
5°7
The seedlings of L. niger emerging from soil Seedlings inoculated with P. sydowiana showed infested with Fusarium equiseti wilted after 10 days very few leaf spots which were black coloured, on and those of V . sesquipedalis showed brown leaf both hosts; the younger leaves were more spots and wrinkled leaf surfaces. Seedling inocula- susceptible. tion of V. sesquipedalis showed no visible symptoms. Severe infection was seen on the leaves of L. niger DISCUSSION seedlings after seedling inoculation, with leaves being badly damaged by large, irregular brown The seeds of L. niger were infested with about 27 lesions with dry centres. fungi and those of V. sesquipedalis with about 22 Though few brown spots were present on some fungi. Among these, Colletotr ichum lindemuthianum, leaves of seedlings of V . sesquipedalis emerging from Ma crophomina phaseolina and Diaporthe phaseosoil infested with F. moniliforme, no other symptoms lorum are the only fungi reported by Noble & were seen on larger seedlings after spraying them Richardson (1968) to be seed-borne pathogens. with the fungus. However, four out of ten seeds of Species of Aspergillus, Penicillium, Mucor, ChoaneL. niger germinated, one of which later wilted. On phora , Syncephalastrum and a few others can be larger seedlings, only minute black spots were seen considered as storage fungi or non-pathogenic on leaves. The fungus infection occurs mostly at fungi, commonly found on the seeds . Other fungi germination but not on larger plants. The fungus recorded here are seed-borne fungi which may be reduces germination, pre-emergence and probably pathogenic to the host plants. Colletotrichum sp., isolated from seeds of V. causes wilting. The leaf spotting in these experiments is of little importance as compared with the sesquipedalis, caused veinal necrosis of both host plants, L. niger being more susceptible. It is reduction in germination and emergence. Fusarium sambucinum was seen to have little recorded as a seed-borne pathogen of L. niger effect on germination of seeds of both hosts. After (N oble & Richardson, 1968) and is parasitic on seedling inoculation with this fungus, only a few other leguminous plants including Vigna sp. leaf spots were seen on V . sesquipedalis but very (C. M .I. distribution Map. No. 177; Sristava & large light-brown lesions with thin but distinct dark Mathur, 1954). It causes anthracnose of pods and brown borders were seen on the leaf margins of seeds of leguminous crops. L. niger. Ma crophomina phaseolina was isolated from seeds Brown leaf spots were seen on both host of V . sesquipedalis and was found to be pathogenic seedlings emerging from soil infested with Macro- to both host seedlings, wounded and unwounded. phomina phaseolina. In seedling infection trial, It is reported on many different host plants, L. niger showed severe leaf spots, portions of stems including Vigna sp., L. niger, is also found in soil and petioles started to rot , and one plant died after and is a seed-borne pathogen of V . sinensis (L utt relJ 8 weeks. Black sclerotia and pycnidia were found & Weimer, 1952; Sackston, 1969 ; Tehon & Bowe, on the surface of stems of the dead portions. One 1939; Wallace, 1932). This fungus causes charcoal other plant ceased to grow ; young leaves were rot, ashy stem blight or stem canker of seedling etiolated and stems rotted, On V . sesquipedalis, no beans and is injurious to seed lings devitalized by leaf spots were seen, but three plants shed leaves other factors (H offmaster, Maclaughlin, Ray & and died 2 months after inoculation. Areas of stem Chester, 1943). Pycnidial formation on diseased were brown in colour and black sclerotia and portions found here, has also been reported by pycnidia formed on the infected surface. Labora- others (Bhargva, 1965; Ghaffar & Kafi, 1965; tory experiments using this fungus on both host Mathur, 1967). seedlings, wounded and non-wounded, showed Diaporthe phaseolorum was isolated from seeds of that this fungus infects some unwounded seedlings L. niger and was previously reported to be a seed-borne pathogen of V. sesquipedalis. As infection but wounding led to almost 100 % infection. As the seedlings emerging from soil infested with studies were not carried out with this fungus, the Nigrospora sacchari were normal and healthy, this pathogenicity of this fungus is not reported. Other fungi parasitizing seeds and seedlings of fungus was not tested in seedling infection studies. Whereas, Phyllostieta sp ., which was not tested in the host plants are Diplodia sp ., Drechslera sativum, soil inoculation studies, promoted severe leaf- Fusarium equiseti, F. moniliforme and Phyllosticta infection symptoms on seedlings of L. niger and sp. Some seedlings emerging from soil infested some seedlings of V. sesquipedalis. Heavily infested with Diplodia sp. were killed, otherwise few leaves were dead and others withered. The spots leafspot symptoms were seen on seedling inocuwere dark brown in colour with chlorotic margins lation ; also wounding encourages infection of the enlarging to about 10-15 mm. Species identification host plants by this fungus. Dr echslera sativum and Phyllosticta sp . caused more severe leaf spot on of this isolate was not made.
508
Mycoflora of hyacinth and long beans
seedlings of L. niger than on V. sesquipedalis. Seedlings of L. niger emerging from soil inoculated with F. equiseti, died and the leaves of seedlings inoculated with the fungi showed leaf spots. F. monilijorme reduced germination of seeds and caused wilting of one of the emerged seedling. The following fungi had very little or no effect on the infection of seeds or seedlings: Curvularia inaequalis, C. lunata, C. senegalensis, Fusarium sambucinum, Chaetomium globosum and Nigrospora sacchari. Most of the fungi tested for pathogenicity, sporulate well in culture except Macrophomina phaseolina. Hence use of whole culture suspension, i.e. fungi plus added nutrients from media, was found to be a more convenient and uniform method. The control plants sprayed with water or sprayed with agar suspension, were normal and healthy. The chloros pretreatment did reduce the incidence of a number of fungi, which may be superficially present on the seeds.
REFERENCES
BHARGVA, S. N. (1965). Studies on charcoal rot of Potatoes. Phytopathologische Zeitschrift 53, 35-54· C.M.!. DISTRIBUTION MAP No. 177. . GHAFFAR, A. & KAFt, A. (1965). Dolichos lablab, a new
pycnidial host of Macrophomina phaseoli (Maubl.) Ashby. Current Science 34, 515. HOFFMASTER, D. E., MACLAUGHLIN, J. M., RAy, W. W. & CHESTER, K. S. (1943). The problem of dry rot caused by Macrophomina phaseoli (Sclerotium bataticoiai. Phytopathology 33, 1113-1114 (Abstr.). KANAPATHIPILLAI, V. S. (1972). The effect of systemic fungicides on U. nuda in barley and P. avenae in oats, together with a study of the fungal flora of seeds of some Malaysian crops. Ph.D. Thesis, Queen's University, Belfast. LUTTRELL, E. S. & WEIMER, J. L. (1952). Macrophomina phaseoli: a stem canker and ashy stem blight of cowpea. Plant Disease Reporter 36, 194-195. MATHUR, S. B. (1967). Sclerotia and pycnidia of Macrophomina phaseoli developed on stems of cowpea, beans (Phaseolusvulgaris) and cotton. Phytopathologische Zeitschrift 58, 137-140. NOBLE, M. & RtCHARDSON, M. J. (1968). An annotated list of seed-borne diseases. Proceedings of International Seed Testing Association 33, 1-191. SACKSTON, W. E. (1969). Sclerotia bataticola on seeds of cowpea, Vigna sinensis. Plant Disease Reporter 53, 438-439. SRISTAVA, H. C. & MATHUR, P. B. (1954). Cold storage of field beans (Dolichos lablab). Indian Journal of Horticulture 11, 125-128. TEHON, L. R. & HOWE, G. H. (1939). Charcoal rot in Illinois. Plant Disease Reporter z3, 312-325. WALLACE, G. B. (1932). Report of the Mycologist. Ann. Rep. Dept. Agric. Tanganyika Territory, 1931 PP· 94-97. (Abstract Review of Applied Mycology u, 201).
(Reviewed for publication 14 May 1981)