PLANT GROWTH RESPONSES TO VESICULAR-ARBUSCULAR MYCORRHIZA XII FIELD INOCULATION RESPONSES OF BARLEY AT TWO SOIL P LEVELS

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1 New Phytol. (1981) 87, PLANT GROWTH RESPONSES TO VESICULAR-ARBUSCULAR MYCORRHIZA XII FIELD INOCULATION RESPONSES OF BARLEY AT TWO SOIL P LEVELS C. CLARKE.-^ND B. MOSSE Soil Microbiology Department, Rothamsted Experimental Station, Harpenden, Herts AL5 IJQ, U.K. {Accepted 25 June 1980) SUMMARY The results are described of a field experiment in which barley was inoculated in the field by placing 20 g of soil containing infected roots below each sowing position. Three VA endophytes were used as inoculants and half the plots were given phosphate before sowing. Growth and infection were recorded twice during the experiment and at harvest. In the plots without added phosphate the fresh wt of ears was doubled by inoculation to approximately 100 g m^^ irrespective of endophytes. Added phosphate increased this more than inoculation and weight of ears rose to 200 to 300 g m"^ With one inoculant dry wt of ears was 35 % greater than with phosphate only, but with the other two it was marginally less. The results are discussed in relation to other experiments with barley and to the levels of infection throughout the experiment. INTRODUCTION Field responses to inoculation with VA mycorrhizal fungi have been shown in maize (Khan, 1972), wheat (Khan, 1975) and barley (Saif and Khan, 1977) in a Pakistan soil containing 15 p.p.m. NaHCOg (Olsen) soluble P. The barley was inoculated before transplanting into the field and yielded nearly four times as much grain as uninoculated plants. With 56 kg P ha~i yield greatly increased in the non-inoculated plants but still remained below that of inoculated, although both had similar shoot dry wts. The best yield was obtained from inoculated plants with added phosphate. The level of Olsen P in plants given superphosphate was not given. Black and Tinker (1979) studied the effects of crop rotations on the development of mycorrhizal infection in barley and on the spore population in the soil. They also surveyed levels of mycorrhizal infection in barley grown commercially, which ranged from 4 to 40 % root length infected, with averages of 14 and 23 % in 2 years. They concluded that infection in barley developed slowly in all rotations and, with some reservations, that final yield was negatively related to mycorrhizal infection. In 1978 the effects of inoculation in situ were examined in a field experiment using onion, lucerne and barley (Owusu-Bennoah and Mosse, 1979). Because the experiment was begun late in the season growth rather than yield responses were obtained. Inoculation stimulated growth of barley in that part of the plot containing 9 mg P (Olsen) kg"i but not in the less deficient part containing 13 mg P (Olsen) kg"^ Growth stimulation resulting from inoculation was much less in barley than in lucerne or onion. Such differences in mycorrhiza dependence of different host species are well authenticated (Baylis, 1972; Cooper, 1975; Johnson, 1976; Menge, Johnson and Platt, 1978; Yost and Fox, 1979). Because barley is X/81/ $02.00/ The New Phytologist

2 696 C. CLARKE AND B. MOSSE an important crop the experiment was repeated in an adjacent site in order to obtain grain yields and to establish at what level of soil-available P the responses of barley to inoculation ceased. Growth and yield of barley inoculated with three different endophytes were compared with those of non-inoculated plants infected only with the indigenous endophytes in the field, in plots with and without added superphosphate. Effects of inoculation on growth and development of mycorrhizal infection were monitored throughout the experiment. MATERIALS AND METHODS Site and experimental design The site (Sawyers I field at Rothamsted Experiment Farm) had been fallowed for 7 years. The soil contained O'l % total nitrogen and 10 mg NaHCOg soluble P(Olsen etal, 1954) kg"' soil (mean of 12 samples). N and Pcontent of soil extracts were measured with a Technicon Autoanalyzer using the methods of Varley (1966) and Fogg and Wilkinson (1958), respectively. The site (18-5 x 12-5 m) was divided into 24 plots of 1 m'^ separated by 0-5 m paths. Before planting, ground agricultural chalk was applied to the site at the rate of 7-5 t ha \ raising the ph in 0-01 M CaCla from 4 to 6-5. Nitro-chalk was added at the rate of 100 kg N ha~' and muriate of potash at the rate of 60 kg K ha~'. Superphosphate was added to half the plots at the rate of 82 6 kg P ha"'. All fertilizers were forked in lightly before sowing. Each plot was sown with washed barley seed (cv. Ark Royal), placed 12 cm apart within and between the rows so that each plot contained 49 plants. Two seeds were placed in each planting position and later thinned to one plant. The plots were watered by hand as required and the plants sprayed twice with Bayleton (Triadimefon) against mildew and three times with Aphox against aphids. There were eight treatments. Three inoculants were compared with uninoculated controls in plots with and without added phosphate. Each treatment was replicated three times in a randomized block design. Inoculum The endophytes used were: (Ij) a mixture of endophytes containing mainly a yellow vacuolate spore type {Glomus mosseae); (I2) a red-brown laminate spore type (G. caledonius) and (I3) a form of G. fasciculatus closely resembling the spore type Eg described by Gilmore (1968) and widely used under that name. The Latin names follow the nomenclature proposed by Gerdemann and Trappe (1974). Ij inoculum, also used in a previous field experiment (Owusu-Bennoah and Mosse, 1979), was originally supplied by Dr F. T. Sanders. These inocula were built up in irradiated soil and sand (1:4) in pots with maize as the host plant. After infection was well established in the maize about 20 g of the infested soil:sand mixture, containing also infected root pieces, was placed 3 cm below the seed in each planting hole. A similar amount of uninfected maize roots in soil and sand was placed below the seeds in the non-inoculated treatments. Records The experiment lasted 14 weeks. Plant size (category 1 to 5) was recorded 3 weeks after emergence for every seedling in each plot. Stems and ears were counted 8 and 10 weeks after emergence. At harvest all stems and leaves were cut off at ground level and the ears separated from the rest. Ear numbers, fresh and dry wt

3 Responses to VA mycorrhizas XII 697 of ears and shoots were recorded. The dried ears were milled and analyzed for total N after Kjeldahl digestion and for total P after igniting with magnesium acetate at 500 C, and dissolving the residue in HCl. N and P content of the extracts were determined as above using a Technicon Autoanalyzer. Root infection was assessed on a representative root sample taken from each plot 3 and 9 weeks after emergence. At harvest roots and surrounding soil to a depth of 8 cm were taken from 10 plants in fixed positions evenly distributed o\er each plot. The samples were bulked and NaHCOg soluble Olsen P was determined for each soil sample. The roots from each plot sample were washed free of soil, cut into 1 cm lengths and well mixed. From each bulk sample 5 g were taken and prepared for microscopic examination by clearing in KOH and staining in Trypan Blue. Percentage root length infected was determined by using the grid line intersect method. A sample of the stained roots was spread evenly over a Petri dish marked with half-inch grid squares as described by Giovannetti and Mosse (1980). Presence or absence of infection was recorded at 200 root: grid line intersect points. More detailed microscope observations were also made on 20 infected root pieces selected from each plot sample to check on the establishment of the introduced fungi. RESULTS P content of soil At harvest, plots without added superphosphate contained from 8 to 11 mg (Olsen) P kg"' soil (mean 10 mg kg~') and plots with superphosphate between 23 and 49 mg P kg"' (mean 39 mg kg"'). Although the amount of superphosphate had been calculated to raise available P to 1 5 mg kg"', based on previous trials in this soil, the desired level was greatly exceeded in the surface layer. The probable explanation is that calculations were based on autumn fertilization and incorporation into the top 22 cm by ploughing, whereas the superphosphate was forked into the top 8 cm just before sowing. As a result the top 8 cm of soil contained much more than intended, while the lower soil layers were probably little affected by the added fertilizer. According to Mattingly and Johnston (1976) barley is unlikely to respond to added phosphate in soils containing more than 20 mg (Olsen) P kg"'. Growth Means for treatments P and PIj are based on only two replicate plots because plants in one replicate of each treatment were yellow and grew abnormally badly, probably because of a large tree root passing below the two plots. Size differences between the uninoculated controls without added phosphate and the other treatments were already apparent 3 weeks after seedling emergence (Table 1). The median category for controls (C) was 1, compared to 3 for the inoculated treatments (I,, L and I.j) and 4 for all 4-P treatments. Results were analyzed using the Mann-Whitney U test. This showed that growth categories were significantly lower for control plots (C) than for all others, and were significantly higher for plots with added phosphate than for those without (F < 0 001). Significance levels for the main factorial effects on subsequent measurements, based on transformed data (logarithms) are given at the bottom of Table 1. As most biologists prefer to see the untransformed data, these are given in Table 1, together with the relevant L.S.D. figures. The results from unamended and +P plots were analyzed separately because the soil was chosen deliberately to show a positive

4 698 C. CLARKE AND B. MOSSE o a \D fn 00 OJ 00 r^ 10 rt- rs '<*! T-H rs O r^ - ' ro. I. ( -+ o rs q^ lo fn -^ O rs ' «^J cr in O fn " t - 'rf- oo c ra cd l-l ID c 1 1 o o ^ C rs. ly-) ( CJ p o x: a. «# # # # # # # # # # * # * c ra I-,' a o ra ij u >-^ d c La OJ 6 c 4-1 o tn 1) =^ u ""^ u l-l 13 O u ^ c o o C C C 3

5 Responses to VA mycorrhizas XII 699 growth response of barley to added phosphate, whereas we particularly wanted to find out responses to mycorrhizal inoculation, especially in +P plots, and the effects of different inoculants. The results show clearly that there was a large response to phosphate, with or without inoculation. This response was evident in all the records taken. Although not always statistically significant it is also evident that in the non-amended plots inoculation approximately doubled all growth parameters measured and that there was little difference between the three inoculants. Essentially this confirms the Table 2. Phosphorus and nitrogen content of ears (percetitage dry ynatter) Unamended Plus superphosphate C I, I. h P PI, PI, PI;. P = 0-05 % p % N C = control; P = 8-2 g P m~^ I, = inoculated with a mixture of endophytes mainly YV (G. mosseae), I = inoculated with G. caledonius, I3 = inoculated with Ej (a form of G. fascicttlatus). previous year's trials (Owusu-Bennoah and Mosse, 1979) although growth increases were then only 33%, but in that experiment the barley was badly affected by mildew. In the +P plots G. caledonius (I^) appeared to be a better inoculant than the other two, supporting its performance last year as a better inoculant for lucerne and onions (Owusu-Bennoah and Mosse, 1979). While I, and I3 were, if anything, growth-depressing compared to the uninoculated plants (P), I, appeared to improve growth and increased dry wt of ears by 35%. Growth depressions, apparently associated with higher levels of mycorrhizal infection, have also been observed in barley by Black and Tinker (1979) and Sanders (pers. comm.). The dry matter content of cars from inoculated plants was greater than that from uninoculated; it ranged from 40 to 43% in unamended and from 49 to 53 % in + P plots compared with 36 and 42 % in the respective controls. It was also greater in ears from +P plots than in those from comparable unamended ones. These figures refer to ears rather than grains because the plants were not fully mature at harvest and the grains were difficult to extract. Phosphorus content of the ears was little affected by treatments (Table 2) but the nitrogen content was slightly lower in the +P treatments (*** by factorial analysis) and highest in uninoculated unamended controls (C). This may be a reflection of the growth limiting effect of phosphate deficiency leading to accumulation of nitrogen in the smaller plants although the percentage P of ears was no lower than in the other treatments. Infection Inoculation greatly increased mycorrhizal infection in the 3-week-old seedlings (Table 3) and added phosphate decreased it. The relatively high level of infection in inoculated plants so soon after emergence shows one important result of inoculation. Infection hy indigenous endophytes was clearly more depressed by added phosphate than that of the introduced endophytes. Although I., (G. caledonius) was the most beneficial of the three inoculants, particularly in +P

6 7OO C. CLARKE AND B. MOSSE treatments, it tended to produce less infection than the other two at most sampling dates. Levels of infection in the unamended control plot (C) were of the same order as those observed in a survey of field soils by Black and Tinker (1979) at comparable sampling dates and those in inoculated plots were similar to those reported by Saif and Khan (1977). Particularly during early growth plants in treatment C were very variable in size and small plants were much less infected Table 3. Effects of inoculation and added phosphorus on length of mycorrhizal root (percentage) Age at sampling (weeks) C Unamended I, I3 P Plus superphosphate PI> PI, PI Significance level for main factorial effects Percentage the Logit analysis of the data confirmed "^' ' following differences: infection Phosphorus Fungus Interaction 3 weeks: C and P < inoculated treatments (/' = 0-05) 3 weeks ** *** li > all other treatments except Ij 9 weeks * PIi > PIj and PL, 14 weeks *** *** 9 weeks: P < all other treatments *** p = 0-001, * P = 0-01, * P = weeks: I, and L, > all other treatments P < all other treatments C = control P = +8-2 g P m~^. 1, = inoculated with a mixture of endophytes mainly YV (G. mosseae), I2 = inoculated with G. caledonius, Ij = inoculated with E;, (a form of G. fasciculatus). Table 4. Percentage of roots with indigenous {fine), introduced and mixed infection. (Mean of 20 1-cm mycorrhizal root pieces from each of three replicate plots) Unamended Plus superphosphate Inoculant Fine Mixed Introduced Fine Mixed Introduced I, Ij I, Ij = a mixture of endophytus mainly YV (G. mosseae), I^ = G. caledonius, L, = E;, (a form of G. fasciculatus). than larger ones. Infection in treatment C increased steadily as new roots encountered fresh propagules in the soil and after 9 weeks percentage infection was similar to that of inoculated plots (Ii_3). The apparent drop in percentage infection in the inoculated plots between the 3- and 9-week assessment probably reflects growth of new roots beyond the inoculum and perhaps inability of the inoculants to keep up with root growth. It does not indicate that the inoculants were dying out or unable to compete with the indigenous endophytes. This is shown by the higher rate of infection in inoculated (Ii_3) than in control (C) plots at the final harvest and by the results summarized in Table 4.

7 Responses to VA mycorrhizas XII 701 The detailed examination of roots under the compound microscope confirmed the good establishment of the introduced endophytes (Table 4). A predominant component of the indigenous endophytes population was a 'fine' endophyte, easily distinguished from the introduced by its small diameter hyphae with nodular s-wellings, proliferation immediately after entry into the root and finely branched arbuscules. The inoculant E., (treatment I3) was further distinguished from the fine endophyte by the production of numerous oval vesicles containing a single large oil vacuole, and the endophyte mixture (treatment I,) by massive de\elopment of external mycelium and many clumped arbuscules. G. caledonius had few additional distinguishing features except its obviously coarse hyphae. That the fine endophyte was an important component of the indigenous population is also confirmed by the relatively high soil infectivity in spite of its low spore count. The figures show an interesting differential interaction between the fine endophyte and the three inoculants. The fine endophyte was strongly suppressed by the mixed inoculum (Ij), retaining only 2% of roots as the sole inhabitant compared with 80 ";, of roots captured by the much more aggressi\'e I,. By contrast the inoculant L, (E.,) competed less well with the fine endophyte and retained only 21 % of roots to itself. Thus the least beneficial endophyte was also the least competitive in both unamended and amended soils. This raises the interesting question whether a beneficial effect on its host has survival value for a VA endophyte. The figures also confirm the preferential suppression of the indigenous fine endophyte by added phosphate which was already shown in Table 3. In +P plots roots infected with only the introduced endophyte far outnumbered those infected solely by the fine endophyte. DISCUSSION In this experiments barley was grown in field plots containing 10 mg (Olsen) P kg"' soil and inoculated at the time of sowing. All three inoculants approximately doubled weight of ears. Superphosphate added to half the plots before sowing raised the Olsen P to 39 mg kg"' but it is likely that this increase remained confined to the top 10 cm of soil. The added P further increased yield by approximately threefold and indigenous infection fell from 45 to 11 "(,. Clearly the plants were able to utilize the added P without appreciable aid of a mycorrhizal system. Nevertheless one endophyte further increased yield by 35",,, but the other two caused marginal yield depressions. Essentially these results confirm those already reported for barley by Saif and Khan (1977). However, the present experiment overcomes two criticisms levelled at the earlier experiments: (1) that of artificiality because the seedlings were transplanted into the field and not sown in situ; and (2) the more serious criticism that the transplanted mycorrhizal and non-mycorrhizal seedlings, although apparently of equal size, may ha\ e been unequal in P content when they were planted out. The apparently unequal growth responses to the three inoculants in - -P plots shows the complexity of factors involved in mycorrhizal associations and the difficulty of predicting inoculation responses. Whether the slight yield depressions due to two of the inoculants (Ij and I.,) are true, and support the apparently negative relationship between percentage infection and yield deduced by Black and Tinker (1979) from rotation experiments in soils containing only 8 to 14 mg (Olsen) P kg"' is uncertain. Their observations were based on indigenous infection rather than inoculation and on much lower infection levels ranging from 5 to 16%. Because of the uneven distribution of the added P in this experiment, the

8 C. CLARKE AND B. MOSSE threshold value of soil P for successful inoculation of barley remains undetermined. Evidently it depends also on amounts and kinds of indigenous endophytes and on the inoculant used. The results illustrate some principles rather than make an assessment of the value of field inoculation for barley. They show (1) that inoculation in the field can greatly increase percentage infection in quite young seedlings, (2) that introduced endophytes can be more tolerant of added fertilizer than indigenous ones, (3) that the eflfects of introduced endophytes can differ and (4) that any beneficial effects do not necessarily relate to the level of infection produced. Although already deduced from various pot experiments (Mosse, 1975, 1977; Abbott and Robson, 1977; Powell and Daniel, 1978), some in sterilized soils, it is interesting to have such findings confirmed in the field. Inadvertently the results of this experiment may illustrate another principle, perhaps suspected but not before formulated: it is this. In a situation of acute P deficiency any introduced endophyte will markedly improve growth, particularly if it leads to early infection. ' Under such conditions the growth increase may be controlled more by available soil P than by endophyte species. However, in a naturally more fertile soil, or one supplied with P fertilizer, an endophyte may need to be more carefully selected for good adaptation to the soil or the host if good responses to field inoculation are to be obtained. ACKNOWLEDGEMENTS We wish to thank Miss S. Smith, C. Calvert and V. Estaun for help in the field. REFERENCES ABMOTT, L. K. & RonsoN, A. D. (1977). CJrowthstimulationof subterranean clover with vesicular-arbuscular mycorrhizas. Australian Journal of Agricultural Research, 28, BAVLIS, C;. T. S. (1972). Minimum levels of available phosphorus for non-mycorrhizal plants. Plant (S Soil, Hi.ACK, R. & 'I'lNKia!, p. B. (1979). The development of endomycorrhizal root systems. IL Effect of agronomic factors and soil conditions on the development of vesicular arbuscular mycorrhizal infection in barley and on the endophyte spore density. New Pliylolofiist, 83, CooPHR, K. M. (1975). Growth responses to the formation of endotrophic mycorrhizas in Solanum, Leptoslicrmum and New Zealand ferns. In: Endomycorrliizas (ed. by F. Iv Sanders, B. Mosse & P. B. Tinker), Academic Press, London and New York. Fooc, D. N. & WILKINSON, N. T. (1958). The colorimetric determination of phosphorus. Analyst, 83, CinnDEMANN, J. W. & TRAPPE, J. M. (1974). The Endogonaceae ii-i the Pacific Northwest. Mycologia Memoir no. 5 pp. 75. CJlLMORii, A. Iv (1968). Phytomycetous mycorrhizal organisms collected by open-pot culture methods. ///feni-(/yn, 39, CJiovANNin'Ti, M. & Mossii, B. (1980). An evaluation of techniques to measure vesicular-arbuscular infection in roots. New Phytologist, 84, JoilN.soN, P. N. (1976). Effects of soil phosphate level and shade on plant growth and mycorrhiza. NeiK Zealand Journal of Botany, 14, KUAN, A. G. (1972). The effect of vesicular-arbuscular mycorrhizal associations on growth of cereals. 1. Effects on maize growth. Nezo Phytotogist, 71, KHAN, A. G. (1975). The effect of VA mycorrhizal associations on growth of cereals. II. Effects on \yheat growth. Annals of applied Biology, 80, MATTINCI.V, CJ. E. C. & JoiiNsroN, A. E. (1976). Long-term rotation experiments at Rothan-isted and Saxniundham Experimental Stations: the effects of treatments on crop yield and soil analyses and recent modifications in purpose and design. Annals of Agronomy, 27, MF.NGE, J. A., JOHNSON, A. L. V. & Pi.A-rr, R. G. (1978). Mycorrhizal dependency of several citrus cultivars under three nutrient regimes. Nezv Phytologist, 81, MoKSE, B. (1975). Specificity in VA mycorrhizas. In: Endomycorrhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker), pp Academic Press, London and New York. Mo.ssE, B. (1977). Plant growth responses to vesicular arbuscular mycorrhizas. X. Responses of 5(v/o.snH(/ifs and maize to inoculation in unsterile soils. Neiv Phytologist, 78,

9 Responses to VA mycorrhizas XII 703 OLSEN, S. T., COLE. C. U., W.ATANABE. F. S. & DEAN, L. A. (1954). Estimation of avaitahlc phosphorus in soils hy extraction with sodium bicarbonate. Circular - United States Department of. \griculture, 939. OWUSU-BENNOAH, E. & MOSSE, B. (1979). Plant growth responses to vesicular-arbuscular mycorrhizas. XI. Field inoculation responses in barley, lucerne and onion. Nezc Phytologist, 83, POWELL, C.LL. & DANIEL, J. (1978). Mycorrhizal fungi stimulate uptake of soluble and insoluble phosphate fertilizer from a phosphate-deficient soil. Nezv Phytolof^ist, 80, SAIF, S. R. & KHAN,.A. G. (1977). The effect of vesicular-arbuscular mycorrhizal associations on growth of cereals. III. Effects on barley growth. Plant & Soil, 47, VARLEV, J. A. (1966). Automatic methods for the determination of nitrogen, phosphorus and potassium in plant material. Analyst, 91, YOST, R. S. & Fox, F. L. (1979). Contribution of mycorrhizae to the P nutrition of crops growing on an Oxisol. Agronomy Journal, 71,

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