Phytopath.2, 113, 311-316 (1985) (D 1985 Verlag Paul PareyI Berlin und Hamburg ISSN 0031-9481 / InterCode: PHYZA3

College of Agriculture, King Saud University, Riyadh, Saudi Arabia

Interaction of Meloidogyne incognita and Macrophomina phaseolina in a Root-Rot Disease Complex

of French Bean

BY AHMAD S. AL-HAZMI

Received April 2, 1984

Abstract Effects of Mcloidogync incognita and Macrophornina phaseolina on root-rot of two cultivur of

French bean were examined in a greenhouse experiment, Severity of Macrophornina root-rot increased by 54.5, 94.6, and 9.6 % when both pathogens were introduced simultaneously, the nematode fmt by 2 weeks, and the fungus fiist by 2 weeks, respectively. Nematode infection and reproduction was adversely affected when the fungus was introduced fmt. Cultivv *Harvester" was more tolerant to both pathogens and less susceptible to the nematode than *Romano Italian".

Zusammenfassung Die Intcraktion von Mdoidogyne incognita und Macrophomina phaseolina

inncrhalb dcs Krankhcitskomplcxcs Wurzclfiule der Gartcnbohne In einem Gewachshausversuch wurden die Auswirkungen von Mcloidogync incvgnitr und

Mucrophornha phascolina auf die Wurzclfiule von zwei Gutenbobensorten untcrtucht. Die Schwere der Mucrophorninu-Wurzelfiule erhohte rich um 54,5,94,6 und 9,6 % wenn beide Pathogene gleichzeitig, der Nematode zuerst fir 2 Wochcn, beziehungsweise der Piz zuerst fur 2 Wochen, eingesetzt wurden. Die Nematodeninfektion und -reproduktion wurdc gegenteilig bccinfludt, nach- dem der Pilz zuerst eingesetzt wurde. Die Sorte .Hanester* war gegenuber beiden Pathogenen toleranter und weniger anfillig gegeniiber dem Nematoden ds die Sorte .Romano Itdim=.

French bean, Phaseolus vulgaris L., is an important vegetable crop in Saudi Arabia, and is susceptible to root-knot nematodes. Meloidogyne incognita (Kofoid and white) Chitwood and Macrophomina phaseolina (Tassi) Goid are often associated simultaneously with ' the roots of French bean under field conditions. Infected plants are usually 'stunted and their root system often show extensive gall development and root rot symptoms.

US. Copyright C lemw Center Code Suumcnti 0031-9481/85/1304-0311$02.50/0

312 AL-HAZMI

The importance of root-knot nematodes in disease complexes has received much attention. Many reports have indicated that combinations of Meloidogyne spp. and root-infecting fungi may result in greater damage than either pathogen acting alone (BATTEN and POWELL 1971, CARTER 1975, CAUQUIL and SHEPERED 1970, MORRELL and BLOOM 1981, PORTER and POWELL 1967, POWELL 1968, POWELL and NUSBAUM 1960). TU and CHENG (1971) reported that incidence and severity of root-rot caused by Macrophomina phaseoli (Maubl.) Ashby (= M. phaseolina) increased in Kenaf seedlings simultaneously infected by Meloidogyne javanica (Treub) Chitwood. They also found that nematode pene- tration increased when both pathogens were present.

The present investigation was carried out to determine the interrelation between Meloidogyne incognita and Macrophomina phaseolina in a root-rot disease complex of two cultivars of French bean.

Materials and Methods Treatments were conducted in a split-plot experiment. The two main plot treatments were two

French bean cultivars, "Harvester* and "Romano Italian'. The six sub-plot treatments included were: 1) M. incognita alone (N); 2) M.phareohna alone 0; 3) both pathogens simultaneously (N + F); 4) nematode fmt and fungus two weeks later (N+ F); 5) fungus fmt and nematode two weeks later (F+ N); and 6)"control (CK). A randomized complete block design with six replicates was used.

Seeds of either cultivar were sown in plastic pots (18 an diameter) containing a steam-sterilized mixture of equal pans of peat, sand and soil and maintained in a greenhouse (2S3O"C). After seed germination, seedlings were thinned to one seedling per pot. Two weeks later, seedlings were inoculated with the nematode, fungus, or both. The second inoculations were made two weeks after the first.

MeZoidogyne incognita was originally collected from infected tomato plants in the field and multiplied on eggplants in the greenhouse. For i n d u m , eggs were extracted with 0.5% sodium

Table 1 Effect of Meloidogyne incognita (N) and Macrophomina phaseolina ( F ) singly and combined upon

root-rot and root-knot of two French bean cultivad

Treatment Root-rot index ( 0 - 5 ) Root-knot index (0-5) Romano Harvester Treatment Romano Harvester Treatment Italian means Italian means

Control 0.00 a N 0.00 a F 1.83b N + F 2.38 b c N+F 3.33 c F+ N 1.83 b

Romano Italian Harvester

0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 3.66 b 1.50b 1.67b 0.00 a 2.33bc 2.58b 2.50 c 3.16~ 325 d 4.16b 1.83b 1.83bc 2.50 c

cultivu means 2.46 a 2.21 a

0.00 a 0.00 a 4.16b 3.91 b 0.00 a 0.00 a 4.33 b 3.42 b 3.66 b 3.91 b 2.33 c 2.42 c

3.21 a 3.62 a

-

'I Data subjected to split-plot analysis; different at P - 0.05; 2 weeks before fungus, F-+ N = fungut applied 2 weeks before nematode.

Means followed by the same letter are not significantly Sequences: N + F = simultaneous inoculation, N-. F = nematode applied

Interaction of Meloidogync incognita and Macrophomina pbareolina 313

hypochlorite (HUSSEY and BARKER 1973). Each pot received'14,OOO eggs in about 30d of distilled water equally distributed around the roots.

Macrophomina phaseolina was originally isolated from infected French bean plants collected from the field. Cultures were maintained, in the laboratory, by frequent transfers to fresh potato- dextrose-agar (PDA). Inoculum was prepared by culturing the isolate in the dark on FDA medium in Petri-plates at 3OoC for 7 days. Cultures were then flodded with sterile distilled water, and sclerotia were scraped from the agar surface with a rubber spatula. The resulting suspension was strained through cheesecloth and sclerotia were counted and adjusted to a concentration of 2 x 1O%nL A fmal inoculum concentration of 500 sclerotidg soil was used. The inoculum suspension for each pot was spread over the soil surface and then covered with a thin layer of sand, Plants were watered regularly and fertilized with Greenzit" NPK (0.3 %) as needed.

Sixty-four days after the first inoculation, the experiment was terminated. Data were recorded on pod weight, root fresh weight, root-knot index, root-rot index, and egg production. Eggs were extracted from roots with 0.5 % sodium hypochlorite for lOmin (HUSSEY and BARKER 1973). Root- knot index was based on a 0.0 to 5.0 scale where: 0.0 = no galls; 1 = 1-2 galls; 2 = 1-10 galls; 3 = 11-100 galls; and 5 = more than 100 galls per root system. Root-rot index was determined by scoring the severity of disease on a scale ranging from 0.0 (no disease) to 5.0 (severe root-rot).

Results Roots of plants inoculated with M-incognita bore many galls with no

significant difference between the two tested cultivars (Table 1). However, when M . phaseolina preceded nematode inoculation by two weeks, gall development was significantly reduced. Root-rot index of plants inoculated with the fungus increased in the presence of the nematode (Table 1). The highest and significant increase of root-rot occurred when the nematode preceded fungal inoculation by two weeks. However; this increase was not significant in the reciprocal treatment (Table 1).

Root weights of inoculated plants were significantly reduced in all treat- ments except one (Table 2). No significant differences were found between cultivars or among treatments. However, visual symptoms of nematode and

Trble 2 Effect of Meloidogyne incognita (N) and Macrophominapharcolina (F) singly and combined upon pod

and root weights of two French bean cultivarsy ~

Treatment Pod weight (g) Root fresh weight (g) Romano Italian Harvester Romano Italian Harvester

Control N F N+P N4F F-, N

Romano Italian Harvester

39.69 a' 34.69 a 55.33 a 11.54b 33.90 a 34.00 b 19.16 b 34.75 a 20.78 b 12.82 b 33.02 a 21.95b 15.75 b 19.12 b 20.60 b 16.77b 28.58 a 27.68 b

cultivar means 18.83 a 30.68 b

49.570 29.13 b 25.42 b 25.71 b 24.81 b 37.13rb

30.06 a 31.96a

~~

I Data subjected to split-plot analysis; different at P - 0.05; 2 weeks before fungus, F* N - fungus applied 2 weeks before nematode.

Means followed by the same letter are not significantly Sequences: N + F simultaneous inoculation, N+F = nematode applied

314 AL-HKLMI

fungal infection were more evident on roots than root weight differences indicate. Roots of plants inoculated simultaneously with both pathogens or with nematode first were short, less dense and fibrous, and became darker due to coalescing lesions. The severity of root-rot was more pronounced when nematode preceded fungal inoculation.

Pod weights of plants inoculated with either or both pathogens were significantly reduced in cv. "Romano Italian" but not in "Harvester" (Table 2). However, when nematode preceded fungal inoculation, pod weight of the, latter cultivar was significantly reduced. Cultivar means show that pod weights of "Harvester" were significantly higher than that of "Romano Italian".

Reproduction of M. incognita on both cultivars was adversely affected when fungus preceded nematode inoculation (Table 3). This suppresion of nematode reproduction was 91 % on "Romano Italian" and 95 % on "Harvester". Simul- taneous inoculation with both pathogens showed a similar effect on "Romano Italian" but not on "Harvester". However; nematode reproduction increased considerably on "Romano Italian" when the fungus was introduced two weeks later. Cultivar means showed that nematode reproduction was significantly less on "Harvester" than on "Romano Italian" (Table 3).

c Ta b l r 3 Effect of Macropbomina pbaseolina (F) on reproduction of Mrlodogyne incognitcr (N) on two

cultivus of French beany

Treatment cultivar Romano I d a n Harvester

mean eggs/g. of root N 4 262 az 5538 a N + P 992 b (76.v 2738 a (50.6) N d F 29 336 c 4354a (21.4) F + N 389 d (90.9) 261 b (95.3)

Romano Italian 8745 a Harvester 3223 b

cultivu means

Data were transformed to log (X + 1) and then subjected to split-plot analysis; Means followed by the same letter are not significantly different at p = D.05; ' Sequences: N + F.P simultaneous inocuh- tion, N-, F = nematode applied 2 weeks before fungus, F-, N P fungus applied 2 weeks before nematode; P percentage change of nematode reproduction.

Discussion It is evident, from this experiment, that hl.phaseolina, normally a weak

pathogen (CHUPP and SHERP 1960), can cause severe root-rot when present with well-established root-knot nematode infection. Severity of root-rot increased by 54.5,94.6, and 9.6 % when both pathogens were introduced simultaneously, the nematode first by 2 weeks, and the fungus first by 2 weeks, respectively. Tu and

Interaction of Meloidogync incognita and Macrophomina phareoha 315

CHENC (1971) reported similar effects of M. javanica and M.phaseolina when simultaneously introduced on Kenaf seedlings. If M . incognita preceded fungal inoculation, by 2 weeks, root-rot was significantly higher than that of the reciprocal, or fungus alone, treatments. In fact, M . phaseolina alone did not cause much root-rot on inoculated plants. M. incognita was, then, capable of enhancing this normally weak pathogen to become a major pathogen. Both pathogens, singly or cbmbined, did not affect pod weight of cv. “Harvester” except when the nematode was introduced first. In this case, plants were predisposed by M. incog- nita and tolerance was lost.

Predisposition of plants by M . incognita to fungal infection was previously noted with fungi other than M.phaseoZina (BATTEN and POWELL 1971, PORTER and POWELL 1967, POWELL 1968). Although, no attempt was made to examine shorter or longer periods of time, two weeks were long enough to cause such an effect. It has been reported that physiological changes in roots with galls provide much of the basis for root-knot nematode-fungus interaction (BAITEN and POWELL 1971). Such modification may change infected root tissues to be more favourable for fungal infection.

On the contrary, infection and reproduction of M. incognita on both cul- tivars were unfavourably affected when the fungus was introduced first. How- ever, Tu and CHENC (1971) reported that M.phaseoZina infection was favourable to the reproduction of M, javanica in Renaf roots when both pathogens were introduced simultaneously on 5,10, and 15 day-old seedlings. The discrepancy in both studies might be due to nematode species, host, or inoculation treatments. POWELL (1971) reported that, in general, populations of migratory nematodes appear to increase as the result of interactions with fungi; whereas, populations of sedentary nematodes are generally suppressed under similar conditions. The suppression of M. incognita reproduction, reported in this study, might be due to the adverse effect of the fungus on the nematode penetration, or because the fungus directly invades the giant cells which disrupts nematode feeding and subsequent reproduction within the roots. Histological changes of infected roots are under investigation.

Literature BATTEN, C. K., and N.T. POWELL, 1971: The Rhuoctonica-Mclojdogyne disease complex in flue-

cured tobacco. J. Nematol. 3,164-169. CARTER, W. W,, 1975: Effects of soil temperature and inofulum levels of Mefoidogync incognira and

Rhizoctonia rolani on seedling disease of cotton. J. Nematol. 7,229-233. CAUQUIL, J., and R. L, SKEPERED, 1970: Effects of root-knot nematode-fungi combinations on

cotton seedling disease. Phytopathology 6 0 , 4 4 ~ ’ 5 1 . CHUPP, C., and A.SHERF, 1960: Diseases and their Control. The Ronald Press Company, New

York. HUSSEY, R.S., and K.R. BARKER, 1973: A comparison of methods of collecting inocula of

Mcfoidogyne rpp., including a new technique. PI. Dis, Rep. 57,1025-1028. MORELL, J. J., and J. R. BLOOM, 1981: Influence of Mcloidogync incognita on Fusuium wilt of

tomato at or below the minimum temperature for wilt development. J. Nematol. 1 3 , 5 7 4 0 . PORTER, D. M., and N. T. POWELL, 1967: Influence of certain Meloidogync species on Fusdum wilt

development in flue-cured tobacco. Phytopathology 57,282-285,

316 AL-HAZMI, Interaction of Meloidogyne incognita

POWELL, N.T., 1968: Disease complexes in tobacco involving interactions between Mel&dogyne incognita and soil-bone fungal pathogens. Proc. First Int Cong. Plant PathoL London. July 1968. -- ,1971: Interactions between nematodes and fungi in disease complexes. Annu. Rev. Phy- topathol. 9,253-274.

--,and C.J. NUSBAUM, 1960: The black Shank-root knot complex in flue-cured tobacco. Phytopathology 50,899-906.

Tu, C. C., and Y. H. CHENG, 1971: Interaction of Mcloidogyne javanicd and Macrophominaphaseoli in Kenaf root rot. J. Nematol. 3 , 3 9 4 2 .

Author's address: AHMAD SMD AL HAZMI, Department of Plant Protection, College of Agriculture, P. 0. Box 2460, Riyadh, 11451 (Saudi-Arabia).