Notes and briefarticles

SPEAKMAN, J. B. & LEWIS, B. G. (1978). Limitation ofGaeurnannornyces grarninis by wheat root responsesto Phialophora radicicola. New Phytologist 80, 373-380.

WARD, E. W. B. (1961). The vitamin requirements ofOphiobolusgrarninis Sacco CanadianJ ournal of M icro-biology 7,423-425.

WARD, E. W. B. & COLOTELLO, N. (1960). The im-portance of inoculum standardization in nutritionalexperiments with fungi. Canadian J ournal of Micro-biology 6, 545-556.

WARD, E. W. B. & HENRY, A. W. (1961). Comparativeresponse of two saprophytic and two parasitic fungito temperature, hydrogen-ion concentration andnutritional factors. Canadian Journal of Botany 39,65-79·

WHITE, N. H. (1941). Physiological studies of thefungus Ophiobolus grarninis Sacco I. Growth factorrequirements. Journal of the Council for ScientificResearch 14, 137-146.

EFFECTS OF PESTICIDES ON MYCORRHIZA INFIELD-GROWN BARLEY, MAIZE AND POTATOES

J. A. OCAMPO* AND D. S. HAYMAN

Soil Microbiology Department, Rothamsted Experimental Station,Harpenden, Herts. AL5 2JQ

Vesicular-arbuscular mycorrhizal (VAM) fungiare common in arable soils and can enhance plantnutrient uptake and growth (Gerdemann, 1975;Hayman, 1978; Mosse & Tinker, 1980). Hencethe value of applying pesticides to field cropsmight be partially counterbalanced by side effectson the VAM fungal population. In glasshouse potexperiments, VAM infections were reduced byvarious pesticides (Hayman, Macdonald & Spokes,1978), including fungicides (N esheim & Linn,1969; Jalali & Domsch, 1975; El-Giahrni, Nicolson& Daft, 1976); benomyl was especially active(Bailey & Safir, 1977; De Bertoldi et al. , 1977;Boatman et al., 1978). Conversely, the nematicideDBCP (i .a-dibromo-j-chloropropane) increasedVAM infection in cotton (Bird, Rich & Glover,1974). With peanuts the nematicide/insecticidecarbofuran reduced mycorrhiza in both pot andfield experiments (Backman & Clark, 1977). VAMpopulations in field-grown potatoes were reducedby the insecticide aldrin and in tomatoes by theinsecticide metasystox (Kruckelmann, 1973), butwere little affected by other pesticides on .fieldbeans (Salt & Hornby, 1971). Spore numbers ofVAM fungi in field-grown peas were affected byfungicides, but not consistently (Stewart &Pfleger, 1977). Soil fumigants have strongereffects ; for example formalin greatly reducedVAM populations in a wheat field (Hayman, 1970)and methyl bromide usually eliminates the VAMpopulation almost completely, for example incitrus nurseries (Kleinschmidt & Gerdernann,1972).

* Present address: Seccion de Microbiologia,Estacion Experimental del Zaidin, Granada, Spain.

The variability of results from arable fieldsprompted us to examine mycorrhizal popula-tions of pesticide-treated plots in field trialsof barley, maize and potatoes at the Rothamsted(clay loam) and Woburn (sandy loam) farms, asfollows:

(1) Barley (H ordeum vulgare L. 'Julia ') in ButtClose, Woburn : benomyl at 22 kg ha-\ dazomet(general biocide) at 224 kg ha-1, aldicarb (pri-marily a nematicide) at 6 kg ha - 1 and controls.All plots had received annually 27 kg ha- 1 of Pand 52 kg ha-1 of K as compound fertilizer andeither 38 (N vr ) or 75 (N -2) kg ha-1 ofN as 'Nitro-chalk' 25.

(2) Barley (' Julia ') in Long Hoos field V,Rothamsted: benomyl at 4 kg ha-\ aldicarb at6 kg ha- 1, chlorfenvinphos (insecticide) at 2 kgha-\ chlortoluron (herbicide) at 2 kg ha- 1 andcontrols . All plots had been initially treated withthe herbicide 2,4-D and given annual dressings of54 kg ha - 1 of P and 205 kg ha>' of K as com-pound fertilizer and 125 kg ha-1 of N as 'Nitro-chalk' 25.

(3) Maize (Z ea mays L. 'Anjou 210') in ButtFurlong, Woburn: dazomet at 450 kg ha-1 andcontrols. All plots received annually 61 kg ha-1 ofP and 232 kg ha- 1 of K as compound fertilizerand 50 kg ha- 1 of N as 'Nitro-chalk' 25.

(4) Maize (' Calderon 535 ') in Long Hoos fieldVI , Rothamsted : benomyl at 11'2 kg ha-1, dazo-met at 450 kg ha- 1, aldicarb at 4'5 kg ha-\phorate (insecticide) at 1'65 kg ha -l, a mixture ofbenomyl +dazornet +phorate at their respectiverates and controls. All plots received annually59 kg ha" of P and 225 kg ha-1 of K as com-

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Notes andbriefarticlespound fertilizer and 50 kg ha? of N as 'Nitro-chalk' 25.

(5) Potatoes (Solanum tuberosum L. 'PentlandCrown') in Great Hill, Woburn: benomyl,thiabendazole (fungicide), and carbofuran, all at22 kg ha-t, and controls. All plots had beengiven N, P and K at 242, 106 and 309 kg ha-1,

respectively, as compound fertilizer.In October 1977, three replicate root-soil

samples were collected from each of two to fourreplicate plots per treatment, depending on theexperiment. Each sample consisted of five bulkedsubsamples taken from the top 10 cm of soilalong the plant rows. After thorough mixing,50 g was wet-sieved (Gerdemann & Nicolson,1963) for counts of VAM spores. A random rootsample of twenty 1 em pieces was examined atx 160 magnification after staining with trypan blue(phillips & Hayman, 1970). VAM infection was

recorded as before (Ocampo, Martin & Hayman,1980). The data were analysed statistically on logtransformations.

Table 1 summarizes the results. Spore popula-tions were rather low compared to numbersgenerally reported for arable soils (see Hayman,1978). The laminate type (Masse & Bowen, 1968),similar to Glomus macrocarpus var. geosporus(Gerdemann & Trappe, 1974) was predominantin all five sites, comprising 70 to 90 % of thetotal population. A few white reticulate andyellow vacuolate types (Mosse & Bowen, 1968)and some unidentified spores were also found.Consistently fewest spores were found in thebenomyl-treated plots. Dazomet also reducedspore numbers, especially in maize, as did phorate.The effects of aldicarb were variable; it had littleeffect on spore populations in barley and maize atRothamsted but increased spore production in

Table 1. Mycorrhizal populations of field crops of barley, maize and potatoestreated with different pesticides

Root Root EntrySpores/loa g length cortex points/em Vesicles/em

Crop/Site Pesticide soil infected infected root root(%) (%)

Barley:Butt Close, N-l None 63- 8- 3"7- 4'S- 6'3-

Benomyl 38b 5- 1"4- 2"2- S"3-Dazomet 46b 4- 1"6- 2"3- 4'0-Aldicarb 94c S8b 46'3b 23"7b 166·8b

Butt Close, N-2 None 43- 3- 1'1- 2'7- o'S-Benomyl 27b < l b o-r> O'2b 0-Dazomet 42- l b 0"2b o-s- 0'7-Aldicarb so- 4- 1'7- 1·8- 4'7b

Long Hoos None S6- 3- 0'9- 1'4- 0Benomyl 3Sb 2-b 0'4- 1'0- 0Aldicarb 49- 3- 0'7- l'S- 0Chlorfenvinphos 79c 3- 0'9- 1'2- 0Chlortoluron 76c l b 0'4- 0"9- 0

Maize:Butt Furlong None 2S- 24- 19'0- 6'9- 0'4'

Dazomet Sb 4b 2'Sb 2"lb 0'2'Long Hoos None 20S' 60- 34'1- 23'0' 7'0'

Benomyl 97bC 29b 17'Sb 15'7'b ObDazomet 136'bc 31b 17'Sb lS'7-b ObAldicarb 167-b 44- 2S'7- 20"0_b l'3-bPhorate 124-bC 27b 14'9b 11'7b ObBenomyl+Dazomet +Phorate lo4c 14c 7'Sc 6'3c O'7b

Potato:Great Hill None 8- 2- 0'S- 1'3- 0

Benomyl 6-b 2- 0,6- 1'3- °Thiabendazole 3b 1- 0"2- 0"7" 0Carbofuran 9' S' 1'1" 3'7" 0

Data analysed on log transformation and t test. Within each group, means differ significantly (P = o'OS)where not followed by the same letter.

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Notes and briefarticles

barley at Woburn. In the potato experiment VAMspores were too sparse for treatment differencesto be evaluated.

Most mycorrhizal infection occurred in maize,especially in the control (no pesticide) plots atLong Hoos field where the percentage of rootlength infected was double that present in plotstreated with benomyl, dazomet or phorate. Thenegative effect of dazomet on mycorrhiza inmaize was even more marked at Woburn (ButtFurlong). Barley at the lower level of N at ButtClose field showed the biggest pesticide treatmenteffect of all the experiments, with VAM infectionin the aldicarb plots reaching more than seventimes that in the controls. In the other experi-ments, however, aldicarb had no significant effectin either increasing or decreasing infection.Benomyl and dazomet generally had a negativeeffect on infection, and phorate significantly re-duced infection at the one site where it wasapplied. Thiabendazole, carbofuran, chlorfenvin-phos and chlortoluron had no obvious effect.Differences between pesticide treatments' weredifficult to evaluate statistically in the potato andbarley (high N) experiments because mycorrhizalinfection was sparse.

Qualitative differences in infection are evidentin vesicle production, with most vesicles beingformed in barley in Woburn soi!. Numbers ofentry points were usually influenced by pesticidesin much the same way as VA infection of the rootcortex was affected.

The greatly increased V.AMinfection associatedwith aldicarb at Butt Close was especially interest-ing. Bird et at. (1974) explained similar observa-tions with DBCP on cotton by suggesting thatthe nematicide might eliminate nematodes com-peting for the same root sites as VAM fungi, orit might directly affect the physiology of thefungus or root. Higher levels of N seemed tooverride the aldicarb effect.

Our findings from the five experiments ex-amined indicate that populations of VA mycor-rhizal fungi in arable fields are affected, not onlyby fungicides and general biocides, but also bynematicides and insecticides. Sometimes theireffects were slight or even positive, but usuallythey decreased mycorrhizal infections and sporenumbers, an effect that occurred most consistentlywith benomy!. Since some crops (e.g. maize) areusually strongly mycorrhizal, and many plantscan grow better with mycorrhiza, the possibilityof plant protection chemicals indirectly affectingcrop yield by their effects on VA mycorrhizashould receive more attention. In our study,

pesticide effects may have been lessened by thepresence of abundant fertilizer because high Nand P levels generally decrease VAM infectionand spore numbers (Hayman, 1970, 1978) andplant responses to mycorrhiza are generallygreatest in soils deficient in phosphate. It is likelythat pesticides could be more deleterious in lessfertilized soils by inhibiting a larger indigenousmycorrhizal population in a situation where itsstimulatory effects on plant nutrient uptake weremore important to the crop.

REFERENCES

BACKMAN, P. A. & CLARK, E. M. (1977). Effect ofcarbofuran and other pesticides on vesicular-arbus-cular mycorrhizae in peanuts. Nematropica 7, 13-17.

BAILEY, J. & SAFIR, G. (1977). Uptake and effect ofbenomyl in mycorrhizal soybean plants. Proceedingsof the American Phytopathological Society 4, 93(Abstr.).

BIRD, G. W., RICH, J. R. & GLOVER, S. U. (1974).Increased endomycorrhizae of cotton roots in soiltreated with nematicides. Phytopathology 64, 48-51.

BOATMAN, N., PAGET, D., HAYMAN, D. S. & MOSSE, B.(1978). Effects of systemic fungicides on vesicular-arbuscular mycorrhizal infection and plant phos-phate uptake. Transactions of the British MycologicalSociety 70, 443-450.

DE BERTOLDI, M., GIOVANNETTI, M., GRISELLI, M.& RAMBELLI, A. (1977). Effects of soil applicationsof benomyl and captan on the growth of onions andthe occurrence of endophytic mycorrhizas andrhizosphere microbes. Annals of Applied Biology 86,111-115·

EL-GIAHMI, A. A., NICOLSON, T. H. & DAFT, M. J.(1976). Effects of fungal toxicants on mycorrhizalmaize. Transactions of the British MycologicalSociety 67,172-173.

GERDEMANN, J. W. (1975). Vesicular-arbuscular my-corrhizae. In The Development and Function of RODts(ed. J. G. Torrey and D. T. Clarkson), pp. 575-591.London: Academic Press.

GERDEMANN, J. W. & NICOLSON, T. H. (1963). Sporesof mycorrhizal Endogone species extracted from soilby wet sieving and decanting. Transactions of theBritish Mycological Society 46, 235-244.

GERDEMANN, J. W. & TRAPPE, J. M. (1974). TheEndogonaceae in the Pacific Northwest. MycologiaMemoir 5, 1--'76.

HAYMAN, D. S. (1970). Endogone spore numbers insoil and vesicular-arbuscular mycorrhiza in wheatas influenced by season and soil treatment. Trans-actions of the British Mycological Society 54, 53-63.

HAYMAN, D. S. (1978). Endomycorrhizae. In Inter-actions between Non-pathogenic Soil Microorganismsand Plants (ed. Y. R. Dommergues and S. V.Krupa), pp, 401-442.

HAYMAN, D. S., MACDONALD, R. M. & SPOKES, J.R.(1978). The effects of pesticides on VA mycorrhiza.Rothamsted Report for 1977, Part 1, p. 238.

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416 Notes and briefarticles

JALALI, B. L. & DOMSCH, K. H. (1975). Effect of effect of certain fungi-toxicants on the formation ofsystemic fungitoxicants on the development of mycorrhizae on corn by Endogone fasciculata and onendotrophic mycorrhiza. In Endomycorrhizas (ed. corn root development. Phytopathology 59, 297-300.F. E. Sanders, B. Mosse and P. B. Tinker), pp. 619- OCAMPO, J. A., MARTIN, J. & HAYMAN, D. S. (1980).626. London: Academic Press. Influence of plant interactions on vesicular-arbus-

KLEINSCHMIDT, G. D. & GERDEMANN, J. W. (1972). cular mycorrhizal infections. 1. Host and non-hostStunting of citrus seedlings in fumigated nursery plants grown together. N ew Phytologist 84, 27-35.soils related to the absence of endomycorrhizae. PHILLIPS, J. M. & HAYMAN, D. S. (1970). ImprovedPhytopathology 62, 1447-1453. procedures for clearing roots and staining parasitic

KRUCKELMANN, H. W. (1973). Die vesikular-arbusku- and vesicular-arbu scular mycorrhizal fungi forHire Mykorrhiza und ihre Beeinflussung in land- rapid assessment of infection. Transactions of thewirtschaftlichen Kulturen. Dissertation, Universitat Br itish Mycological Society 55, 158-161,Braunschweig. 55 pp, SALT, G. A. & HORNBY, D. (1971). Root rot and wilt

MaSSE, B. & BOWEN, G. D. (1968). A key to the of field beans ( Vicia faba ). Proceedings of the 6threcognition of some Endogene spore types. Trans- British Insecticides and Fungicides Conf erence, 251-

actions of the British MycologicalSociety 51, 469-483. 257.MaSSE, B. & TINKER, P. B. (1980). Effects of mycor- STEWART, E. L. & PFLEGER, F. L. (1977). Influence of

rhizas on nutrition of higher plants. CR C Handbook fungicides on extant Glomus species in associationof Nutrition and Food (in the Press). with field-grown peas. Transactions of the British

NESHEIM, O. N. & LINN, M. B. (1969). Deleterious Mycological Society 69, 318-319.

METHODOLOGY OF A CHITIN ASSAY

J. M. WHIPPS AND D. H. LEWIS

Botany Department, The University, Sheffield S10 2TN

The estimation of fungal tissue in infected plantmaterial is difficult because complete separationand subsequent analysis is often impossible.Histological techniques have been used to obtainreliable estimations but these are usually time-consuming (e.g. Matta & Dimond, 1963; Warnock& Preece, 1971; Clifford, 1972). This has led tothe widespread usage of a chemical estimation offungal biomass in infected tissues by colorimetricassay of hexosamines formed by hydrolysis ofchitin (Sharma, Fisher & Webster, 1977, andreferences therein). One of these techniques (Ride& Drysdale, 1972) has the advantages of beingsimple, rapid and sensitive. The conditions usedto treat chitin (120 % KOH for 1 h at 130 °C)yield a group of compounds (chitosan) and do notcompletely hydrolyse the polymer. Although thetechnique is therefore only an estimate of thetotal chitin present, it is consistent and reliable.We have been unsuccessful in obtaining an in-creased yield of hexosamine using a range ofconcentrations and heating times using trifluoro-acetic acid, now widely used for hydrolysis ofplant cell walls (Jones & Albersheim, 1972;Haselwandter & Whipps, unpubl.).

In the method of Ride & Drysdale (1972), in-fected plant material was first homogenized inacetone and centrifuged and washed with waterbefore hydrolysis with KOH and subsequent

determination of hexosamine. To quantify theamount of fungal tissue present, conversion factors(CF) are required to relate yield of glucosamine tounit dry weight of mycelium. These were obtainedby homogenizing axenically-grown fungus inwater, removing suitable amounts, centrifugingthese to precipitate mycelia, discarding super-natants and subsequently carrying out the KOH-hydrolysis and analysis of hexosamine. The ace-tone extraction was omitted. Similar extractionand analys is have been used by Losel & Lewis(1974), Khan & Strange (1975), Langcake &Drysdale (1975), Langcake & Wickins (1975), andZak (1976).

To assess the importance of this acetone ex-traction, cultures of Fusarium oxysporum Schlecht.ex Fr. inoculated with agar discs (5 mm diam) cutfrom the edge of a rapidly growing Petri dishculture, were grown in 25 ml Vogel's 'N' mineralsalts medium containing 2 %sucrose (Vogel, 1956)and incubated as stationary cultures at 25°. Atintervals, mycelium from 3-5 cultures was col-lected separately on Buchner funnels, washedwith water and homogenized in water (20 ml) attop speed of an MSE homogenizer for 1 min.Six samples (0'1 ml or 0'3 ml) from each culturewere transferred to 8 ml Pyrex Sovirel tubes.Three acted as controls and three were washedonce with acetone (5 ml) for 20 min with shaking

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