Proc. Indian Acad. Sci. (Plaat Sci.), Vol. 95, No. 1, August 1985, pp. 35--40. 9 Printed in India. Response of groundnut (Arachis hypogaea L) to combined inoculation with Glomus mosseae and Rhizobium sp K PARVATHI, K VENKATESWARLU and A S RAO Botany Department, Nagarjuna University, Nagarjunanagar 522 510, India MS received 20 September 1984; revised 19 April 1985 Abstrar The adequate level of inoculum of a vesicular-arbuscular mycorrhizal fungus, Glomus mosseae, required for copious mycorrhizal formation in groundnut (Arachis hypogaea L) in ste soil was found to be one gram dried root powder from infected plants per kg soil and this level was u~jzcl subsequently in an inoculation study. In two cultivars of groundnut, combined inoculation with a Rhizobium sp and G. rnosseae resulted in synergistic effect on the formation of mycorrhiza and nodulation when compared to separate inoculations. The combined inoculum resulted in fairly goocl response of the plants in terms of dry weight, uptake of nitrogen and phosphorus. Keyword.s. Glomus mosseae; groundnut (Arachis hypogaea L); Rhizobium sp; vesiculararbuscular mycorrhtza. 1. Introduction Inoculation ofdifferent crop plants with VA mycorrhizal fungi and Rhizobium was found to have synergistic bene effect on nodulation, nitrogen fixation and plant growth (Crush 1974; Daft and E1-Giahmi 1976; Mosse et al 1976; Bagyaraj et al 1979). This increase was attributed to better phosphorus nutritien of legumes as a result of mycorrhizal formation. Smith and Daft (1977) showed that time is an important factor in the development of the tripartite symbiosis between lucerne, Rhizobium meliloti and G. mosseae. Asimi et al (1980) observed considerable decrease in infection by G. mosseae in soybeans at higher levels of phosphate (0-5 or 1 gkh2pojkg ). Groundnut was shown to forro mycorrhiza with Glomus mosseae (Nic. & Gerd.) Gerd. & Trappe in this locality (Rao and Parvathi 1982). The present paper reports the effect of inoculants of Glomus mosseae and Rhizobium sp on mycorrhizal formation and nodulation in two cultivars of groundnut. Initially, the inoculum potential of the VAM fungus for its maximum development in groundnut was also ascertained. 2. Materials and methods The soil used in all expe was a phosphorus deficient (P, 0-0045 ~o) latosol with a ph of 8-1. The potting medium consisted of a 2: 1 soil: sand mixture taken in 20-cm earthen pots and autoclaved. In order to determine the inoculum potential of Glomus mosseae, different levels of inoculum (0-05, 0-1, 0-2, 0.4, 0:6, 0-8, 1.0, 2-0, 4-0 and 6.0 g kg-1 potting medium) in the forro of dried and powdered groundnut roots colonized by G. mosseae were thoroughly mixed. Unsterilized and uninoculated potting mixture served as control. For each inoculum level three pots were maintained and seeds of groundnut cv TMV-2 were sown. The pots were maintained in an open 35
36 K Parvathi, K Venkateswarlu and A S Rao shade at a temperature range of 27 to 31 ~ After 60 and 100 days ofgrowth, five plants were removed for each inoculum level. The roots were cleared with Korl and stained with trypan blue (Phillips and Hayman 1970) and percentage root length infection as well as the number of vesicles or spores cm-i root were determined, in a random sample of 25 to 50 one cm pieces, by the root-slide technique (-Nicolson 1959). At the last sampling, plant dry weight and pod number were estimated for each inoculum level. Nitrogen and phosphorus in plant tissues were determined by the methods of Jackson (1967) and Fogg and Wilkinson (1958), respectively. The data on per cent root infection and the number of vesicles/spores were statistically analysed as detailed earlier (Parvathi et al 1985). In another experiment, a soil-sand mixture was taken in 20-cm earthen pots and sterilized. Seeds of groundnut (cvs TMV-2 and Robut 33-1) were inoculated with peat culture of Rhizobium sp (1 g of peat/100 seeds) obtained through the courtesy of Dr P T C Nambiar, ICRISAa', Hyderabad, and sown in pots containing sterilized soilsand mixture. The number of viable cells was 109 g- I peat culture. The inoculum of G. mosseae in the forro of dried infected groundnut roots (0"1 ~o w/v) was added to the potting medium, ten days after the addition of Rhizobium sp, which provided the following combinations: (a) uninoculated control, (b) G. mosseae alone, (c) Rhizobium sp alone and (d) Rhizobium sp + G. mosseae. For each combination, three pots with 10 seeds sown in each pot were maintained. After 60 and 90 days of inoculation, five plants were taken out from the three pots and the number and weight of nodules per plant, the per cent root infection and the number of vesicles or spores cm- 1 root length were determined. The plant dry weight, nitrogen and phosphorus content were estimated in triplicate samples and the values are represented as averages. 3. Results A high level of mycorrhizal development was observed in the plants raised in unsterilized uninoculated potting medium (table I). The plants raised in sterilized potting medium with inoculum levels of zero to 0.1 g (kg-i soil) failed to develop mycorrhiza. There was a gradual increase in mycorrhizal infection at inoculum levels of 0.2 g to 1.0 g. Beyond this no further significant increase occurred (figure 1). A comparison of results (table 1 and figure 1) shows that there was very little change in per cent infection between 60 and 100 days. By 60 days, infection in the root systems reached a plateau, at higher levels of inoculum. However, sporulation by the fungus increased significantly at 4.0 and 6.0 g levels of inoculum. The plant dry weight, estimated 100 days after inoculation, was greater at the inoculum level of I g than at lower levels (0 to 0-8 g). The growth response was almost the same at higher levels as at 1.0 g level. The number of pods formed was more (5 pods plant-:) with 1.0 g inoculum than at the lower tevels (0 to 0-8 g) which varied on the average from 1-8 to 3.6 per plant. There was no further increase in pod number at the higher inoculum levels (2, 4 and 6 g) when compared to 1"0 g level. The per cent of nitrogen and phosphorus was higher in the plants raised with 1.0 to 6.0 g inoculum than at lower levels of inoculum. The results on the effect of inoculation with Rhizobium and G. mosseae on plant growth, nodulation and phosphorus content in the two cultivars of groundnut are given in table 2. In the cv TMV-2, there was no nodulation at all in uninoculated control plants and in the plants inoculated only with G. mosseae. Plants inoculated
Glomus mosseae and Rhizobium in 9roundnut 37 Ttble 1. Response of groundnut plants (cv TMV-2) raised in sterilized soil supplemented with different levels of root powder inoculum of G. mosseae. VA mycorrhiza, after 60 days of plant growth Plant analysis, after 100 days of growth Inoculum level Vesicles or (g powdered Root length spores N P Pod roots kg- ] soil) infected (~o) cm- 1 root (~ (~o) number 0 0 0 2"72 0"5 1"8 0"05 0 0 2"96 0-5 2.0 0.10 0 0 3"13 0"6 2.0 0-20 2(Y'* 1-2.* 3.32 0"6 2-9 0.40 34* 3"0 3"62 0-7 3.0 0-60 36* 4.0 4-22 0-8 3-2 0"80 42 5-0 4.56 0.9 3-6 1.00 56 6-2* 5.19 1-2 5"0 2-00 64 "6.9 t 5"19 1.2 5.0 4-00 68 8-7** 5'14 1"3 5.2 6-00 74 12-4** 5-13 1-4 5.0 Unste 58 4.2 4.98 0-8 4.0 uninoculated * o* Significantly different from the unsterilized, uninoculated at the 95 ~o and 99 ~o confidente levels, respectively. Zero values are highly significant. I00.2 f~ 2.5 80 6O f.s ~- a: x_ t~ ~20 0.5 q,' ~ ~ INOCuLUM, g/kg SOll- d Figure 1. Impact of VA mycorrhizal inoculum (dried root powder) levels on i. plant dry matter production and b. per cent root infection in groundnut, after 100 days of plant growth.
38 K Parvathi, K Venkateswarlu and A S Rao Table 2. Response of groundnut plants to inoculants of Rhizobium sp and G. mosseae. VA mycorrhiza Nodules Plant analysis Inoculum Root Average Days length Vesicles Dry wt dry after infected or spores Number ptant- i matter N P sowing (%) cm- t root plant- I (mg) (g/plant) (~) (~) (a) cv TMV-2 Uninoculated 60 0 0 0 0 0-66 3-98 0"6 90 0 0 0 0 1-05 4.10-0.8 Glomus mosseae 60 32 2"6 0 0 0-93 4"48 1-1 90 44 3"9 0 0 1"32 5"73 1"7 Rhizobium sp 60 0 0 23.0 5.0 1-04 5-15 1" I 90 0 0 25.0 6.0 1 "63 5"93 I "5 Rhizobium sp + 60 48* 4.2 ~ 41.5 10-0 2.60 5.68 1.8 Glomus mosseae 90 60 ~ 5.8 ~ 57.0 12.0 3.41 6-29 2"7 (b) cv Robut 33-1 Uninoculated 60 0 0 0 0 1-02 4-37 0-7 90 0 0 0 0 1.49 5-19 1-0 Glomus mosseae 60 32 2.9 0 0 1"29 5-10 1.4 90 48 4.2 0 0 2.10 5.57 1.9 Rhizobium sp 60 0 0 44.0 20.0 1-46 5-62 1-4 90 0 0 42.0 20-0 2-25 6-24 1.7 Rhizobiura sp + 60 64 ~ 5.8 ~ 85-0 37-5 3.15 6-20 2.6 Glomus mosseae 90 72" 6"6" 88.0 36-0 3.94 6-73 3-3 Significantly different (P< 0-05) from the corresponding values with G. mosseae inoculation alone. with Rhizobium sp nodulated well. The number, size and dry weight of nodules in the plants inoculated with G. mosseae plus Rhizobium were greater than in the plants inoculated with Rhizobium sp alone, at 60 and 90 days after inoculation. Thus, in combined inoculations there were 57 nodules per plant as against 25 nodules when Rhizobium sp alone was used as the inoculum, at 90 days of plant growth. The uninoculated control plants and the plants inoculated with only Rhizobium sp did not show any mycorrhizal infection. The plants receiving the combined inoculum ofrhizobium sp and G. mosseae gave significantly (P < 0.05) high ievels ofcolonization of roots and sporulation by the mycorrhizal fungus, when compared with the plants receiving only the mycorrhizal fungus. The dry weight and the per cent of nitrogen and phosphorus were higher in plants raised with Rhizobium sp and G. mosseae than in separate inoculations or in uninoculated plants. This increase was most pronounced when the plants were 90 days old. Almost similar results were obtained with cv Robut 33-1 and the effects ofcombined inoculum were even greater in this variety when compared with TMV-2 cultivar (table 2). 4. Discussion Although the relationship between the plant and the fungus in VAM formation is symbiotic, the primary process in the establishment of the fungus in roots is infection.
Glomus mosseae and Rhizobium in 9roundnut 39 For maximal infectivity, sutiicient fungal inoculum must be present in the soil. The inoculum potential of G. mosseae in the present study was assessed by using different quantities of powdered roots from plants showing heavy infection. The results clearly show that 1 g dried root inoculum kg- l soil was optimal for mycorrhizal formation in groundnut by G. mosseae which almost equailed the level of infection in unste soil. Furthermore, the data indicate that dry matter production was closely associated with infection, both reaching a plateau beyond 1.0 g inoculum kg-x soil. Likewise, Carling et al (1979) reported an increase in the infection units (entry points) in young soybean seedlings with increasing quantities of Glomusfasciculatus inoculum, until a maximum was reached. Smith and Walker (1981) while presenting a mathematical model of the infection also established a similar relationship between inoculum and infection of roots of Trifolium subterraneum by VA mycorrhizal fungi. The present investigation also brings out clearly that VA mycorrhiza greatly enhanced nodulation in groundnut plants inoculated with Rhizobium sp. The relatively lower response ofplants at 60 days of inoculation with Rhizobium sp or Rhizobium sp + G. mosseae or G. mosseae only when compared to 90 days suggests that the crop derived greater benefit from nitrogen fixed as well as from P taken up in later stages of plant growth. These findings ate in close agreement with those of Mosse (1977) on Stylosanthes sp in pot culture experiments using unsterilized soil and those reported by Schenck and Hinson (1973) for dually infected soybeans. Mosse (1977) also regarded that the principal effect of mycorrhizas on nodulation is phosphate-mediated. References Asimi S V, Gianinazzi P and GianinaT-zi S 1980 Influence of increasing soil phosphorus levels on interactions between VA mycorrhiza and Rhizobium in soybeans; Can. J. Bot. 58 2200-2205 Bagyaraj D J, Manjunath A and Patil R B 1979 lnteraction between a VAM and Rhizobium and their effects on soybean in the field; New Phytol. 82 141-145 Carling D E, Brown H F and Brown R A 1979 Colonization rates and growth responses of soybean plants infected by vesicular-arbuscular mycorrhizal fungi; Can. J. Bot. 57 1769-1772 Crush J R 1974 Plant growth responses to vesicular-arbuscular mycorrhiza. Vil. Growth and nodulation of some herbage legumes; New Phytot. 73 743-749 Daft M J and El-Giahmi A A 1976 Studies on nodulated and rnycorrhizal peanuts; Ann. Appl. Biol. 83 273-276 Fogg D N and Wilkinson N T 1958 The colorimetric determination of phosphorus; Analyst (London) 83 406--414 Jackson M L 1967 Soil chemical analysis (New Delhi: Prentice Hall) Mosse B 1977 Plant growth responses to VA mycorrhiza. X. Responses of Stylosanthes and maize to inoculation in unsterile soils; New Phytol. 78 277-288 Mos.se B, Powell C L and Hayman D S 1976 Plant growth responses to VAM. IX. Interactions between VAM, rock phosphate and symbiotic nitrogen fixation; New Phytol. 76 331-342 Nicolson T H 1959 Mycorrhiza in the Gramineae. I. Vesicular-arbuscular endophytes, with special referente to the external phase; Trans. Br. Mycol. Soc. 42 421-438 Parvathi K, Venkateswarlu K and Rao A S 1985 Effects of pesticides on development of Glomus mosseae in groundnut; Trans. Br. Mycol. Soc. 84 29-33 Phillips J M and Hayman D S 1970 Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection; Trans. Br, Mycol. Soc. 55 158-161 Rao A S and Parvathi K 1982 Development of vesicular-arbuscular mycorrhiza in groundnut and other hosts; Plant Soil 66 133-137
40 K Parvathi, K Venkateswarlu and A S Rao Schenck N C and Hinson K 1973 Response of nodulating and non-nodulating soybeans; Aoron. J. 65 849-850 Smith S E and Daft H J 1977 Interactions between growth, phosphate content and nitrogen fixation in mycorrhizal and non-mycorrhizal Medicago sativa; Aust. J. Plant Physiol. 4 403-413 Smith S E and Walker N A 1981 A quantitative study of mycorrhizal infection in Trifolium: Separate determination of the rates of infection and of mycelial growth; New Phytol. 89 225-240