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Agriculture and. Enuironment, l (797 4) 2b9-264o Elsevier Scientific Publishing Company, Amsterdam Printed in The Netherlands

RELATIVE SUSCEPTIBILITY OF VARIOUS PROPAGULES OFEUSAR]UM OXYSPORUM F. SP. M.OI,ONIS TO CHLOROPICRIN UNDERCONDITIONS CONTROLLED BY A NEW LABORATORY APPARATUS

J.P. GOYAL, H. MARAITE and J.A. MEYER

Uniuersitt Catholique de Louuain, Laboratoire de Phytopathologie et d.e Mycologie g6n6roleHeuerlee (Belgium)

(Received September 28th, 1973)

ABSTRACT

Goyal, J.P,, Maraite, H. and Meyer, J.A., 19?4. Relative susceptibility of various propagulesof Fusarium ox.ysporum f. sp. melonis to chloropic n under conditions controlled bv anew laboratoif/ apparatus. Agric. Enuirontu., l : 2b9-264.

An apparatus has been devised to study the relative susceptibility of different soil fungiand their propagules to different fumigants under varying conditions such as soil moisture,soil temperature and soil type. The apparatus makes it possible to expose the fungus for adesirable period to a constant, chosen and measurable concentration of fumigant, undercontrolled conditions and continuous flow.

The principle of operation involves a simple mixing of fresh afu and of chloropicdn-saturated air flowing through two separate capillaries, and the exposure of the sample tothe mkture for a certain time. The concentration of chloropicrin is maintained by passingthe chloropicrin-saturated air through a condenser at a given temperature. The flow rateand air pressure of the system are legulated by capillaries atrd water manorneten. Thechloropi&in is absorbed in isopropyt alcohol and its concentration determined bycolorimetric measutement of the nitrite ion released froh the chloropicrin by decom-position in hot alkaline peroxide.

The test fungus Fasarium oxyspotum f. sp. melonis, was inoculated in soil having thedesired moisture content and the soil was exposed to a chloropicrin mixture for differenttimes. Dilution plate counts on peptone PCNB agar medium were used for populationestimations. The relative susceptibility of different propagules was found in the order ofmycelium, conidia and then chlamydospores.

INTRODUCTlON

The results obtained with soil fumigants against soil-borne pathogens havebeen highly variable in the field and greenhouses and sometimes a $eat failure(Allen and Raski, 1950). Soil types, unsuitable soil moisture ard temperatureacting on the dynamics of the soil fumigation (Van Assche, 1971), and thedifferentiation of the pathogen in more resistant survival forms were amongthe factors determined. For the application of fumigants under optimal

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conditions, a precise knowledge of the incidence of every factor influencingthe efficacy of fumigants is necessary. Therefore, an apparatus has beendesigned which allows the exposure of fungal propagules or soil samples, fordesirable periods, under controlled conditions of temperatrue and humidity toa stdctly. constant and continuous flow of fumiqants with chosen and measur-able concentrations.

MATERIALS AND METHODS

Apparatus

- The assembled apparatus represented by Fig.1 has been designed for thefumigants used in liquid form, where condensation of the vapois is possible,e.g. chloropicdn and methyl bromide. Air is supplied by an air cylinder fittedwith a double-stage pressure reducer. The pressure is further adjusted and keptconstant by a glass tube immersed to a chosen length (maximum 40 cm) in awater column (inlet pressure regulator) allowing the escape of the excess air(Bartholomew and Broadbent, 1949). The air is then humiclifed by passingover the surface of water in two humidifying chambers each b0 cm long and2.5 cm in diameter (Wilson, 1921), and saturated with chloropicrin bybubbling in a wash-bottle (500 ml) equipped with a fritted glass plate(porosity 40 90 p) and padially filled with chloropicrln. A water bath main_tains the temperature in the evaporation unit. The concentration of chloro-picrin in the air is adjusted and kept constant at saturation bv means of a con-denser (C, ) at a temperature at Least 5'C lower than the evaporation unit.

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Chloropicrin condensation at the bottom of the condenser and the temperatureat the outlet of the condenser confirm the stabilised working conditions of theapparatus. The pressure inside the system is measured by a manometer (Mr ).The chloropicrin concentrations in the air can thus be calculated for a givenau presSure on the basis of the vapor pressure of chloropicrir.r at differenttemperatures (Weast et ai., 1966).

In order to reduce the concentration of chloropicrin without reducing thetemperature, fresh air is introduced at the same temperature as the chloropicrinvapor and mixed with the latter at the outlet of the condenser (Cr ). Theproportion of the dilution of the chloropicrin vapor is regulated by capillarytubes (Ct1 and Ctr) placed in the air flow. The mixture is passed in thefumigation chamber (FC), volume 1 100 cm3, through a ftitted glass plate(diameter 9 cm and porosity 90 150 p) fitted for uniform diffusion andbetter contact with the samples. The top of the fumigation chamber is easilyremovable for the manipulations to be made. The sides are covered with a coitfor consta.nt temperature at the time of exposing the samples. At the ou et,the chloropicrin is trapped in isopropanol by passing in wash-bottles (each500 ml) fitted with fritted glass plat€s (porosity 150-200 p). The quantity ofchloropicrin trapped in isopropanol after a desirable time is measured by themethod of Feinsilver and Oberst (1953). This is based upon the colorimetricmeasurement of the nitrite ions released from the chloropicrin by decomposi_tion in a hot alkaline peroxide medium. The ou et pressure can be adjusted bymoving the tube up and down in wash-bot e 2. The flow rate throush thesystem with a set of capillary tubes is regulated by the difference of pressuresat the inlet and outlet, measured by a manometer (Mr). Total flow ii measuredby the time needed to displace a given volume of water from an immersedgraduated cylinder positioned at the outlet. A flow of around 15 ml/sec, per-.mits the fumigation apparatus to be equilibrated in 3 min.

The temperature of the two condensers and the fumigation chamber isregulated by a Lauda KaR Cryostat (range -50 to + 1S0"Ct. The pVC tubesconnecting the various parts of the apparatus are resistant to chloropicrinvapors. The apparatus is operated in a well-ventilated hood. AI1 ihe joints fromwhich leakage of chloropicrin is possible, are carefully checked and silicongrease is applied.

Fungus

, Conidia of Fusurium oxysporum f. sp. melonis (Leach and Currence)Snyder and Hansen, M15 D (Meyer and Maraite, 1971) were hanested byfilhation from 4-day-old shaken culture in MM medium (Meyer, 19b9), washedthree times by centrifugation and resuspended in sterile distilled water.

Mycelium was produced by incubating the washed conidia in MM medium(shaken culture) or in autoclaved soit for 12-14 h at 2b.C.

Chlamydospores were produced by inoculating washed conidia in soilextract supplemented with glucose (Goyal ei al., 1923).

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Propagules were mixed well with greenhouse organic soil (organic mattercontent 7 .2O7o, water holding capacity on dry weight basis 487o and pH 6.4)and a humidity of 507o of the water holding capacity was maintained. Thehoculated soil was exposed in the fumigation chamber at Z5'C for differenttimes. 10 g (on dry weight basis) of soil samples were taken, after each expo-sure and suwival of the fungus was determined by the dilution plate counts onPCNB agar medium (Nash and Snyder, 1962). Contrllls were exposed to a pureair stream for the longest exposure period. The results were expressed as thepercentage of surwival compared to the control.

RESULTS AND DISCUSSION

Under our set conditions, the apparatus has been operated very stably,grvrng a constant flow of air (total flow 13.58 1 0.0b ml/sec) and a constantconcentration of chloropicrin (2.5 mg/min). The quantity of soil samplesexposed in the fumigation chamber was 100 g (on dry weight basis). Nochange in humidity of soil samples was observed during treatments.

Fig.2 very clearly indicates that there is a great difference in the susceptibilityof different propagules of ,F. oxysporum f. sp. melonis. The mycelium is themost susceptible form, followed by conidia and chlamydospores. In our condi-tions, it is possible to kill about 907o mycelium in 12 min, while conidia andchlamydospores require about 52 and 94 min, respectively. Thus tr seems tobe very interesting to induce germination of the survival form by the applica-tion of some energy sources before fumigation, for a better control of thedisease. Cook and Flentje (1967) and Papavizas et al. (1968) could germinate

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Fig.2. Relative susceptibility of mycelium, conidia and chlamydospores of tr'. oxysporumI s-p. melonrs to chloropicrin in soil at b0% of the water holding capacity and exposed at25" C.

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the chlamydospores of formae speciales pisi and phaseolt of F. solnni in soil byapplying some readily available carbon and nitrogen sources.

For the time being the apparatus has been devised only for small-scalescreening tests, but keepirg in view its principles, it is possible to modify it toa large-scale, which may be useful for greenhouse and pot cultwes.

ACKNO\

LEDC EMEN ' f

The senior author is very grateful to the Ur'iversit6 Catholique de Louvainfor awarding a post-graduate scholarship and thanks Mr J. Bourmanne for hisskillful assistance.

REFERENCES

Allen, M.W. and Raski, D.J., 1950. The eflect of soi l type on the dispersion of soi lfumigants. Phytopathology, 40: 1043 1053.

Bartholomew, W.V. and Broadbent, F.E., 1949. Apparatus for control of moisture,temperature and air composition in microbiologieal respiration experiments. Proc. SoilSci. Soc. Amer., 14: 156 160.

Cook, R.J. and Flentje, N.T., 1967. Chlamydospore germination and germling zurvival ofFusarium solani f. pisi in soil as affected by soil water and pea seed exudation. Phyto-pathology, 57 | 774-182.

Feinsi lver, L. and Oberst, F.W., 1953. Microdetennination of chloropicrin vapor in air.Analyt. Chem., 25: 820-427.

Goyal, J.P., Maraite, H. and Meyer, J.A., 1973. Abundant production of chlamydospores byFusarium oxysporum f. sp. melonis in soi l extract with glucose. Neth. J. Plant Path., ?9:162-164

Meyer, J.A., 1959. Moisissures du sol et des l i t iEres de 1a r6gion de Yangambi (Congo Belge).LN .E .A .C . Pub l . , 56 r . Sc i . , No . 75 ,211pp .

Meyer, J.A. and Maraite, H., 1971. Mult iple infect ion and symptom mit igation in vascularwil t diseases. Trans. Bri t . mycol. Soc., 57: 371 377.

Nash, S. and Snyder, W.C., 1962. Quanti tat ive estimation of propagules of the bean root rotFasorium in f ield soi ls by plate counts. Phytopathology, S2t 567 572.

Papavizas, G.C., Adams, P.B. and Lewis, J.A., 1968. Survival of root- infect ing fungi in soi i .V. Saprophyl ic mult ipl icat ion of Fusdrium solani L sp. phaseoli in soi l . Phytopathology,58 : 414 -420 .

Van Assche, C., 1971. Behaviour and perspectives of chemical soi l fumigatign. Proc. 6thBrit . Insect. Fungic. Conf., 706-7 14.

lveast, R.C., Selby, S.M. and Hodgman, C.D., 1966. Hand book of Chemistry and Physics.Chemical Rubber Co., Ohio, pp. D 105.

Wilson, R.E., 1921. Humidity control by means of sul lur ic acid solut ion with cI i t ical com-pi lat ion of vapor pressure data. Indian Eng. Chem., 13: 326-331.

DISCUSSION

Question (Klein, L.): Can you explain why in your laboratory experiment,when adding moisture to the fumigation chamber the rate of kill became lessand more chloropicrin had to added, when under field conditions we recom-

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mend a relatively high moisture content?

Ansruer: Probably high humiclitv restricts the Ultll"P:^ti:ntoopicrin rn

the soiL. For that reason *" """lui u lo'-tg"t-"*pottlt" tT:,I:: our apparatus'

In vour field conditions, no*"u"'' o '"tutliuely high humidity may be necessary

to Lheck the Ioss of fumigants'

Question (Krihun,J ): What was the concentration of chloropicrin in the

air mixture?

Answer'-The concentration of chloropicrin ""d"L:^lt ':::

conditions was

3.0 mg/l air and the rate ol au;;;;hrough the syst€m was 815 ml/mut'

Question (Hoffmann'G ): How have you tested the gerrnination rate of

chlamYdosPores?

Answer: Thechlamvdospores were produced ln :o1t ::tl1----------------:i medium (Goval

et a1., 1e?3), separated l'"'" ;;;;i;;;td.incubltedlllutrient medium at

25"C. After !2-74 h,"tt"tot"o'pi"'oUt"rvations were made We observed up to

947o germination'

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