New Phytol. (1981) 87, 3 5 5.161 355 OMPONENTS OF VA MYORRHIZAL INOULUM AND THEIR EFFETS ON GROWTH OF ONION BY A. MANJUNATH AND D. J. BAGYARAJ Depart?nent of Agricultural Microbiology, University of Agricultural Sciences, G.K.V.K. ampus, Bangalore-560065, India {Accepted 10 May 1980) S U M MARY Studies were conducted to assess the effect of different components of VA mycorrhizal inoculum on growth of onion. The associated micro-organisms and sterilized root segments, did not significantly influence the growth and phosphorus nutrition of onion. Application of mycorrhizal inoculum containing extramatrical chlamydospores and infected root segments containing mycelium, vesicles and arbuscules had a significant effect on shoot and root weight, and phosphorus content of the plant. Further fractionation of inoculum indicated that chlamydospores are the important component, since plants inoculated with chlamydospores produced more dry matter and contained more phosphorus. INTRODUTION The endomycorrhizal inoculum used usually contains hyphae, vesicles and arbuscules in the root pieces and mostly chlamydospores in the soil. Addition of root pieces may add a little organic matter and this may influence plant growth to some extent. In addition to the VA mycorrhizal fungus the inoculum may also contain other soil micro-organisms. There is very little information as to how the different components of the mycorrhizal inoculum infiuence the plant growth and phosphorus (P) nutrition (Hall, 1976). Hence the present study was conducted. In the first experiment the influence of (1) associated micro-organisms (2) sterilized mycorrhizal inoculum and (3) mycorrhizal inoculum usually used, was studied. In the second experiment the effect of (1) root pieces containing arbuscules, vesicles and mycelium and (2) the extramatrical chlamydospores on the growth and P nutrition of onion was studied. MATERIALS AND METHODS Soil Soil used was phosphate deficient (3 mg kg"' of available P by NHjF-t-Hl extraction) sandy loam soil of ph 56 with an indigenous spore population of 52 spores per 50 ml of soil. Pots of 20 cm diameter were filled with 3-5 kg of soil. For sterilization pots were autoclaved at 121 for 1 h. Inoculum Sand: soil mixture containing extramatrical chlamydospores and infected root segments of Sorghimi bicolour var. sudanense infected with Glomus fasiculatus (Thaxt) Gerd. and Trappe and grown for 90 days served as the inoculum. Inoculum contained 750 chlamydospores per 100 g. 0()28-646X/81/020355+ 07 S02.00/0 l'^^' 'l"he ^'' "' Phytologist
356 A. MANJUNATH AND D. J. BAGYARAJ s : Experiment I The treatments included were (a) control; (b) sterilized inoculum; (c) sterilized inoculum with associated micro-organisms and (d) unsterile inoculum. Sterilization of inoculum was done by autoclaving at 121 for 1 h. P'or obtaining associated micro-organisms, 100 g of inoculum was mixed with 100 ml sterile water and the suspension passed through a 45-//m sieve. The leachate flowing out of the sieve containing the associated micro-organisms was collected and used. In all tbere were eight treatments, four under each of sterilized and unsterile conditions. There were six replicated pots for each treatment and each pot contained three seedlings. s: Experiment 2 The treatments included were (a) control; (b) spores + sterilized infected root segments; (c) sterilized spores + infected root segments and (d) sterilized spores + sterilized infected root segments + associated micro-organisms. Sterilization was done by autoclaving at 121 for 20 min. Root segments were obtained by sieving 100 g of inoculum (root + soil) through a 1-mm sieve and picking up the root pieces and washing them well with water. Spore suspension was obtained by wet sieving and decantation method of Gerdemann and Nicolson (1963). Associated micro-organisms were obtained as mentioned in I^xperiment 1. There were eight treatments, four under each of sterilized and unsterile conditions. There were three replications and each pot contained three seedlings. Plants Seeds of onion {Allium cepa var. chikkaballapur red) were sown directly in pots and covered witb a thin layer of the same soil. No nutrients were added. Pots were maintained under greenhouse conditions. In the first experiment plant samples from three replicate pots were removed on the 50th day and remaining plants were removed on the 80th day. In experiment 2, plants were removed 70 days after sowing. Measurements Height of plants was recorded at three intervals in Experiment 1 and four intervals in Experiment 2. Dry wt of shoots and roots were recorded after drying samples to a constant weight at 70. Endomycorrhizal spore numbers were determined by wet sieving and the decantation method outlined by Gerdemann and Nicolson (1963). Infection of roots by VA-mycorrhizal fungi was assessed after staining with trypan blue (Phillips and Hayman, 1970). Phosphorus content was analyzed by vanadomolybdate phosphoric yellow colour method outlined by Jackson (1973). RESULTS Experiment 1 The mean height of onion plants in different treatments is given in Table 1. Plants grown in unsterile soil were taller. Application of mycorrhizal inoculum had a significant effect on height of onion plants at all intervals. Uninoculated control plants and those inoculated witb sterilized inoculum and sterilized inoculum- associated micro-organisms did not differ significantly. In sterilized soil, plants inoculated with mycorrhizal inoculum were taller.
VA mycorrhizal inoculum 357 Table 1. Mean height of onion plants inoculated zvith sterilized inoculum, associated micro-organisms and unsterile uioculutn Height (cm IVeatment 40 d 60 d i) Main effects SI SI+AM UI i) Interactions x X SI X Sl +.AM xui X x SI X SI +.-\M x Ul 16-0 21-8 2-6 17-0 15-9 15-1 27-9 3-7 11-6 11-3 12-3 29-0 22-4 20-4 17-8 26-6 5-3 19-4 28-8 3-2 21-0 20-7 20-0 34-6 4-5 13-4 12-5 14-2 37-5 28-7 28-9 25-9 31-7 6-4 19-9 31-5 3-1 22-8 22-5 22-2 35-1 4-4 14-3 13-1 14-4 37-9 31-3 32-0 29-9 32-7 6-3. \M, associated micro-organisms;, control; d, days after sowing; SI, sterilized mycorrhizal inoculum; sterilized soil; Ul. unsterile m\'corrhizal moculum;, unsterile soil. Table 2. Onion shoot and root dry zvt as influenced by inoculation with sterilized inoculum, associated micro-organisms atid unsterile inoculum Dry wt perplant (mg) Shoot Root 80 d 80 d (i) Main effects SI SI + AM UI D at 1",, (ii) Interactions x xsl x SI + AM xui X SI xsi+.am xui D at I " 103 5 150 0 7()-5 11-1 73-7 285-1 97-4 23-4 30-0 40-4 320-2 117-7 125-5 107-0 249-9 137-7 459-7 845-6 183-0 452-1 471-3 453-0 1234-3 258-0 143-2 143-2 134-5 1418-2 761-0 799-5 771-5 1050-5 366-0 56-7 46-7 25-3 28-8 31-3 121-3 22-0 13-3 25-4 29-3 158-8 37-3 32-2 33-4 83-8 31-1 202-5 311-4 18-3 170-9 166-4 187-3 503-2 25-8 69-3 42-8 74-4 623-5 272-6 289-9 300-2 382-8 36-5.\y\, associated micro-organisms;, control; d, days after sowing;, not significant; SI, sterilized lvcorrhizal inoculum;, sterile soil; Ul, unsterile mycorrhizal inoculum;, unsterile soil.
358 A. MANJUNATH AND D. J. BAGYARAJ Table 3. Phosphorus content of shoot and root as influenced by inoculation with sterilized inoculum, associated micro-organisms and unsterile inoculum P content per plant (y/g) Shoot Root 80 d 80 d (i) Main effects SI SI + AM Ul D at 1 "/ (ii) Interactions x xsi x SI+AM xul xsi X SI+AM xui D at 1 "/ 100 1 139-8 63-8 67-3 70-0 278-6 88-9 23-3 29-0 36-3 311-6 104-3 105-6 103-7 245-6 130-5 272-5 504-0 44-1 290-8 292-7 260-1 709-4 62-4 129-3 68-6 77-0 815-1 452-3 516-8 443-1 603-8 88-3 44-1 36-2 n.k. 19-8 22-3 24-1 94-3 13-8 10-6 20-3 22-2 123-4 29-0 24-3 26-0 65-3 19-6 86-5 118-8 13-4 60-4 77-3 70-3 202-6 19-0 27-1 39-3 27-0 252-6 93-6 115-3 113-6 152-6 26-9 AM, associated micro-organisms;, control; d, days after sowing;, not significant; SI, sterilized mycorrhizal inoculum;, sterilized soil;, unsterile soil; Ul, unsterile mycorrhizal inoculum. Table 4. Glomus fasciculatus spores in soil as influenced by inoculation with sterilized inoculum, associated micro-organisms and unsterile inoculum Spores per 50 ml soil X SI X SI + AM xui + UI D at 1 % 52 60 56 104 88 7-9 80 d 103 98 93 171 159 64-7 Spores were absent in treatments, SI and SI + AM under sterilized conditions, hence analyzed as one w-ay classification. AM, associated micro-organisms;, control; d, days after sowing; SI, sterilized mycorrhizal inoculum;, sterilized soil; UI, unsterile mycorrhizal inoculum;, unsterile soil. Table 2 shows the mean dry wt of shoots and roots. Plants grown under unsterile conditions weighed more. Dry wts of plants inoculated with mycorrhizal inoculum were greater. ontrols, plants inoculated with sterilized inoculum and sterilized inoculum + associated micro-organisms did not differ significantly from one another. In sterilized soil, plants inoculated with mycorrhizal inoculum weighed nearly ten times more than plants of other treatments. Mycorrhizal inoculation caused significant increase in the root weight under unsterile conditions.
VA tnycorrhizal inoculum 359 The P content of shoots and roots is given in Table 3. The P content of plants grown in unsterile soil was more but the differences were significant only at second sampling. The P content of plants inoculated with mycorrhizal inoculum was more. Inoculation of sterilized inoculum either with or without associated microorganisms did not have a significant effect on P content of plants. All the root segments observed were infected with VA-mycorrhizal fungi in unsterile soil. In sterilized soil, the root samples collected from uninoculated plants and those inoculated with sterilized inoculum with or without associated micro-organisms were not infected with VA-mycorrhizal fungi. Endomycorrhizal spore numbers were higher in soil samples treated with mycorrhizal inoculum (Table 4). Table 5. Mean height of onion plants as influenced by inoculation zvith spores, infected root segments and associated micro-organisms Height (cm) 40 d 60 d 70 d (i) Main effects SP IS AM D at 1 % (ii) Interactions x X SP X IS X AM X SP X IS X AM 110 15 2 21 10-7 13-7 15-5 12 7 3 0 8 7 12 3 13-4 9-9 12 7 15 2 17-7 15 5 15 9 19 3 1-8 15 8 22-5 18-6 13-6 2 3 13 9 18-8 18 2 129 17 7 26 3 191 14 3 3-6 17-7 23-2 3-5 17 5 25-7 22-2 16 3 49 14 5 21 8 21 0 13 3 20 5 29-6 23 3 193 19 8 25-7 3-5 19 9 281 25-7 17 5 5 0 15-0 24-8 246 14-9 24-7 31 3 26 8 20-1 AM, associated micro-organisms + sterilized spores + sterilized infected root segments;, control; d, days after sowing; IS, infected root segments4-sterilized spores;, not significant; SP, spores + sterilized root segments;, sterilized soil;, unsterile soil. Experiment 2 The mean height of onion plants is given in Table 5. At all intervals, plants grown under unsterile conditions were taller. ontrols and plants inoculated with associated micro-organisms were shorter. At ays, plants grown under unsterile conditions inoculated with spores were significantly taller than those inoculated with infected root segments. Mean dry wt and P content of plants are presented in Table 6. Plants grown under unsterile conditions weighed more and contained more phosphorus. Shoot and root dry wt and P content of plants inoculated with spores were greater. Inoculation with infected root segments also influenced the dry wt and P content of root significantly, when compared to control and plants inoculated with associated micro-organisms. Inoculation with spores caused significant increase in
360 A. MANJUNATH AND D. J. BAGYARAJ Table 6. Onion shoot and root weight and P content of shoot and root as influenced by inoculation with.spores, infected root segments and associated micro-organisms Di-y wt per plant (mg) P per content plant (/(n) Shoot Root Shoot Root (i) Main eftects D at 1",, SP IS AM (ii) Interactions x xsp x IS x AM xsp x IS X AM D at 1 % 274-0 435-6 110-9 226-3 593-0 339-4 260-5 156-9 131-6 438-2 370-9 155-0 320-9 747-8 307-8 365-9 221-7 99-7 131-0 56-0 195-5 136-6 73-3 30-3 46-6 146-9 163-1 42-2 65-4 244-1 110-2 104-4 42-8 120-6 174-2 35-1 90-9 253-9 140-7 104-2 49-6 53-6 208-6 158-3 62-0 128-3 299-1 123-1 146-3 70-7 51-4 49-5 27-3 75-8 70-7 28-1 17-5 23-3 76-3 84-3 21-9 31-3 75-3 57-2 34-3 24-7 AM, associated micro-organisms + sterilized spores + sterilized infected root segments;. control; d, days after sowing; IS, infected root segments + sterilized spores;, not significant; SP, spores + sterilized infected root segments;, sterilized soil;, unsterile soil. Table 7. Glomus fasciculatus spores in soil as influenced by inoculation with spores, infected root segments and associated micro-organisms Spores per 50 ml soil 110 X SP 236 x IS 135 X AM 93 xsp 170 x IS 149 D at 1 '; 46 Spores were absent in soil samples from pots receiving and AM treatments under sterilized conditions, hence analyzed as one way classification. AM, associated micro-organisms 4-sterilized spores4-sterilized infected root segments;, control; IS, infected root segments + sterilized spores; SP, spores 4- sterilized infected root segn-ients;, sterilized soil;, unsterile soil. dry wt and P content of root in unsterile soil but in sterilized soil it was on a par with infected root segments. All the root segments observed were infected with VA-mycorrhizal fungi in unsterile soil. In sterilized soil, the root samples collected from uninoculated plants and those inoculated with associated organisms were not infected with VA-mycorrhizal fungi. In unsterile soil, inoculation with spores alone increased
VA mycorrhizal iiioeulum 361 the number of chlamydospores significantly. In sterilized soil inoculation with spores and infected root segments had similar effect on mycorrhizal spore numbers (Table 7). L) 1 s c u s s I o N It is evident fiom the first experiment that the micro-organisms associated with mycorrhizal inoculum, even in the presence of sterilized root pieces, have no significant influence on the growth of onion both under sterilized and unsterile conditions. I^^urther, it may be concluded that the beneficial effect of mycorrhizal inoculation on plant grcnvth was only due to the presence of viable mycorrhizal fungus present in form of spores and extramatrical hyphae in soil and infected root segments containing m\celium, arbuscules and \esicles. The response obtained under unsterile conditions upholds the practical significance of inoculation in soils of low phosphate availability suggested earlier by Hayman and Mosse (1971). In the second experiment, the effect of the different components of mycorrhizal inoculum viz. (1) spores; (2) infected root segments containing hyphae, vesicles and arbuscules; (3) associated micro-organisms, on growth of onion was assessed. Plants inoculated with spores grew better and contained more phosphorus. Studies conducted earlier ha\ e shown that the germ tubes emerging from spores produce fan like expanded infection structures with several root penetration points in contrast to single infection point produced by infected root segment (Powell, 1976). Therefore, it might be expeeted that mycorrhizal establishment would be better when the spores are used for inoculation. Under sterilized conditions infected root segments were equally efficient in causing root infection and stimulating the plant growth. Perhaps the sterilized soil in which native microfiora have been destroyed is not very favourable for germination of VA mycorrhizal spores. This view is supported by the earlier observations that the germination of mycorrhizal spores and infection of roots are favoured by certain bacteria, and in unsterile soil (Mosse, 1962; Powell, 1976). In practical agriculture the mycorrhizal inoculum has to be applied to the unsterile field soil. The present study brings out that extramatrical chlamydospores would be an important component of mycorrhizal inoculum. Transportation of large quantities of inoculum is one of the handicaps in the practical field application of mycorrhiza. This could be overcome by wet sieving the mycorrhizal inoculum and collecting only the spores, the volume of which could then be easily handled for field application. REFERENES GERDEMANN, J. W. & Niroi.soN. T. H, (1463). Spores of mycorrhizal Eitdogune species extracted from soil by wet sieving and decanting. Transactions of the British Mycotogical Society, 46, 235. H.AI.i., I. R. (1976). Response of afirosmti rnhusta tc) different forms of endomycorrhizal inoculum. Transactions of the British Mvcohigical Society, 67, 409. H.-\YMAN, D. S. & Mo.ssi:, B. (1971). Plant growth responses to vesicular-arbuscular mycorrhizae. II. In unsterilized soils. The Xezv Phytologist, 70, 29. JAK.SON, M. L. (1973). Soil hemical analysis. Prentice Hall, New Delhi. MOS.SE, B. (1962). The establishment of vesicular arbuscular mycorrhiza under aseptic conditions. J'o//rH(// of General Microbiology, 27, 509. PHII.LII'S, J. M. & HAVMAN, D. S. (1970). Improved procedures for clearing and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society, 55, 158. POWELL,. (1976). Development of mycorrhizal infection from Endogone spores and infected root segments. Transactions of the British Mycological Society, 66, 439.