Zentralbl. Mikrobiol. 145 (1990),23-29

VEB Gustav Fischer Verlag lena

[Istituto di Microbiologia Agraria dell'Universita di Pisa, 1) Centro di Studio per la Microbiologia del Suolo, CNR,Pisa, Italy]

Effect of Soil Temperature on Infective Capacity of Fusarium oxysporumf. sp. dianthi in Presence of Poplar Bark Compost

CARLO FILIPPI and ANTONIO PERA I)

With 4 Figures

Key words: Fusarium pathogenicity, greenhouse carnation plants, different soil temperature, sterilized and notsterilized compost amendments, important soil microbial groups

Summary

The effect of soil temperature on infective ability of Fusarium oxysporum f. sp. dianthi against carnation plantscultivated in a soil amended with poplar bark compost has been investigated.

The compost has been divided in two parts: one sterilized and the other one not sterilized. Subsequently, those twokinds of compost have been added to the same soil on three greenhouse benches. The experimentation has been carriedout by maintaining the soil of each bench at a different temperature, namely 16, 23, 30°C. Temperature and humidityof ambient-air in the greenhouse have been maintained everywhere at the same values.

Variations of most important soil microbial groups have been analyzed and visual readings of plant mortality havebeen taken on 15 days intervals.

It has been shown that soil temperature has no appreciable effect on Fusarium pathogenicity. On the contrary, aless incidence of the disease has been observed in amended soils (both with sterilized and not sterilized compost), withrespect to control soils.

This occurrence has been more evident in soil amended with sterile compost, at increasing temperatures.Likely, nutrients added by compost has caused a response of zymogenic soil-microflora, which is responsible of

the protection against disease. Consequently, the soil temperature influenced the incidence of the disease, simple byacting as a selective factor controlling the growing of microbial soil population.

Zusammenfassung

Untersucht wurde der EinfluB der Bodentemperatur auf die Infektionsfahigkeit des Fusarium oxysporum f. sp.dianthi auf Nelkenpflanzen, die in mit Pappelrindenkompost angereicherten Boden kultiviert wurden. Zur Anwen­dung kam sterilisierter und nicht sterilisierter Kompost in denselben Boden, in 3 Gewachshausbanken. Der Versuchlief unter verschiedenen Bodentemperaturen: 16, 23 und 30°C. Temperatur und Luftfeuchtigkeit im Gewachshauswurde konstant gehalten. Aile 15 Tage erfolgte eine visuelle Bonitur der Pflanzenmortalitat und eine Erfassung derwichtigsten Gruppen von Bodenmikroorganismen. Es stellten sich heraus, daB die Bodentemperatur keinenbetrachtlichen EinfluB auf die Fusariumpathogenitiit ausiibte. Ein geringer Effekt zeigte sich in Boden mit Kompost,verglichen zum Kontrollboden ohne Kompostzusatz.

Am deutlichsten trat dies bei steigender Bodentemperatur in Boden mit sterilisiertem Kompost auf. Wahrschein­lich tragen die Nahrstoffe, die mit dem Kompost dem Boden zugefiihrt werden, zur Entwicklung der zymogenenBodenmikroflora bei, welche ihrerseits vor Krankheitsausbreitung schiitzen. Folglich hat die Bodentemperatur einenEinfluB auf das Auftreten der Krankheit, da sie als selektiver Faktor des Wachstum der mikrobiellen Bodenflorakontrolliert.

Vascular disease caused by Fusarium sp. affect many species of trees and horticultural andfloricultural plants.

24 C. FILIPPI and A. PERA

This pathogen has been becoming more and more virulent and it is in constant expansion inseveralgeographicareas, even if manychemicaland physicalagentshavebeenemployedagainst itin massive quantities.

Vascularfusariosis, causedby Fusarium oxysporum f. sp. dianthi, has beenbeing, since almosttwenty years, the most serious and harmful disease of carnations in Italian cultural conditions.

It is well knownby carnationprocedures that temperature has sensibleeffect on disease causedby Fusarium oxysporum f. sp. dianthi, symptomathology is in fact very evident during late-springand summer periods and less evident and progressively disappearing with incoming autumn(ACCATI and GARIBALDI 1972).

Besides chemical and physical means, several biological means, like root bacterization(ALDRICH and BAKER 1970; BROADBENT et al. 1971; KOTS and GUNNER 1967; MICHAEL andNELSON 1972), soil solarization (COOK et al. 1987; DAVIS and SORENSEN 1986; MARTYN andHARTZ 1986) and suppressive-soil use (ALABOUVETTE et al. 1979; BAKER and COOK 1982; COOK1982; HORNBY 1983), have been experimented against this disease.

Duringlast ten yearsalso the useof compostshasbeenexperimented with thesameaim (DAFT etal. 1979; HOITINK 1980; HOITINK and KUTER 1984; LUMSDEN et al. 1983; MILLNER et al. 1982;VAN ASSCHE and UYTTERBROECK 1981).

Workersof our Institutehave been studying, since a long time, the use of antagonistic bacteriaand of compost against radical phytopaties (FILIPPI et al. 1984; FILIPPI et al. 1985; FILIPPI et al.1987; PERA andFILIPPI 1987).The compostwereobtainedfromwastematerialscomingfromwoodindustries and used as organic amendants.

During an experimentation (FILIPPI and PERA 1988, personal communication) it has beenobserved that, by amending with a compostobtained from poplar bark a soil naturally infected byFusarium oxysporum f. sp. dianthi, carnation protection varied significantly if the trial wasconducted in late-spring or in autumnal period.

Subsequently, FILIPPI and PERA (1989), by amending soil with sterile or non-sterile compost,showedthat a less frequent occurrenceof diseasecausedby Fusarium oxysporum f. sp. dianthi wasimputable to zymogenfractionof soil and not to the microflorawhich was present in the compost.

In the presentwork are reportedthe results relativeto an experimentation by which we intendedto study the protectiveresponseof a soil naturallyinfectedwithFusarium oxysporum f. sp. dianthi,by exposure at three temperatures and amended with poplar bark compost.

The experimentation was carried out at constant humidity and temperature of ambient-air.Visualobservation of plant mortalityand countsof most importantsoil microbial groups have beenmade.

Materials and Methods

Chemical and physical characteristics of compost

Those properties of compost, obtained from poplar bark, are described elsewhere (PERA and FILIPPI 1987; FILIPPIand PERA 1989).

Greenhouse trials

Three benches were made, with dimensions of 1.5 x 4.0 x 0 .3mand were filled with a soil naturally infested byFusarium oxysporum f. sp. dianthi (3000 units/g), which had been taken off from a field in Sanremo (Liguria, Italy) . Inthis country the above mentioned disease is endemic . The benches were maintained at temperatures respectively of 16,23,30 DC. The first temperature (16 DC) was maintained by insufflating into the soil cold and humid air (Fig. 1); theother temperatures were obtained by warming the soil by electrical resistances. Soil temperature control was effectedby thermocouples, with an absolute error not exceeding 1.5 DC. Ambient-air temperature (25 DC) and ambient-airhumidity (70 %) control were effected by a centralized conditioner.

Each bench has been divided into 9 parcels of the same area (about 0.5m2) . Three of those parcels were used as acontrol , 3 amended (20 % w/w) with compost and 3 amended (20 % w/w) with sterilized compost.

Infective Capacity of Fusarium oxysporum f. sp. dianthi 25

~ -~--- --..-....- .-...--- -- ------_...,'.

Fig. I. Scheme of soil thennostatation system (16°C): CD soil@glasswool@grid@distributor(plexiglass)@air­conditioner.

In each parcel 60 rooted carnation cuttings of the cultivar "Olga" were planted out.Visual readings of occurred infection have been extirpated to execute histological tests and verify the nature of

etiologic agent (DIMOCK 1948).

Microbial analysis

Soil samples were taken off in several points of each parcel, at various depths and subsequently mixed. Microbialanalysis was performed twice on each mixed sample. Counts were performed to determine the number of soilmicroorganisms (bacteria, fungi and actinomycetes) by the dilution technique, by using respectively the followingmedia: Nutrient Agar (NA), Potato Dextrose Agar (PDA) (with 50ppm tetracycline hydrocloride added) andWaksman Agar ( NaCI 5gil, glucose 10gil, Bacto Casitone 5gil (Difco), beef extract 3gil (Liebig), Agar 20g/1 (Difco).

Cellulolytic microorganisms (fungi and bacteria) were determined by Hudson (1972) method, which doesn'tmake use of antibiotics.

Microorganisms which exhibited an antagonistic activity toward Fusarium oxysporum f. sp. dianthi weredetermined by a technique reported by FILIPPI et al. (1984).

The number of Fusarium oxysporum f. sp. dianthi propagules was determined according to BOUHOT and ROUXEL(1971) method.

Readings were made after incubation in a thermostat at the same temperatures of soils from which they had beentaken off.

Results

Effects of soil temperature on Fusarium oxysporum f. sp. dianthi induced pathology

Mortality counts performed at several times on carnation plants cultivated in control parcels, at16, 23 and 30°C exhibited non appreciable differences: so that it should be concluded that thevarations of soil temperature does not affect fungus pathogenicity. On the contrary, a less incidenceof pathology with increasing temperature in the soils amended with sterile or non-sterile compostwas observed. This effect is greater in the soil amended with sterile compost.

The patterns of fusariosis infection in the soil are reported in Fig. 2.

26 C. FILIPPI and A. PERA

100

80 T=16'C It60 I ~'S /0//

i 40 01·/!20 I ~~od::, , , ! ,

o 15 30 45 60 75 90

time (days) 2

AT=30'C

oo/.~•

./ o~\~. \.

o

T=23'C

0 ......

t-:~~~e • e .~ _

T=1S'C8

4

S

8B

o 0 15 30 46 60 75 gQ 0 t ~ 46 iRs 15 §'o b ~ ~ 46 60 ~ !fotime ,days I 3

Infective Capacity of Fusarium oxysporum f. sp. dianthi 27

8

6

T=23 ·c A

1----'------'. • • C' •

B

c

t ime (days)

Fig. 4 . Effect of soil temperature on soil microbial population (number of colonies expressed as the quotient betweenthe vlue for parcels amended with compost and control parcels at each sampling time and at the same temperature ).Cellulolyti c microorganisms (A), microorganisms antagonistic to Fusarium oxysporum f. sp . dianthi (B), Fusariumoxysporum f. sp. dianthi (C) . 0--0 sterile compost amended soil; ..-. non sterile compost-amended soil.

Effects of soil temperature on soil microflora

Results of microbial counts pertinent to the several parcels at the above mentioned temperatureswere expressed as the quotient between the value read in parcels amended with compost and thevalue read in control parcels, both taken at the same drawing time and at the same temperature. Theinherent graphics are reported in Fig. 3 and 4.

It can be noted that population of some microbial groups of telluric microflora (bacteria, fungi,actinomycetes and cellulolytic microorganisms) increases with increasing temperature. This effectis greater in the soil amended with sterile compost.

No similar effect has been noted for pathogen antagonists and for pathogen itself.

Fig. 2. Effect of soil temperature on mortali ty of plants . Direct observation of percentage mortality: e-----e plantsdead in control, non-amended soil;0--0 plants dead in non sterile compost-amended soil; j.--j. plants dead insterile compost-amended soil.

Fig. 3. Effect of soil temperature on soil microbial population (number of colonies expressed as the quotient betweenthe value for parcels amended with compost and control parcels at each sampling time and the same temperature ).Bacteria (A), actinomycetes (B), fungi (C) . 0--0 sterile compost-amended soil; e-----e non sterile compost­amended soil.

28 C. FILIPPI and A. PERA

Discussion

The above mentioned results have been obtained with constant values of ambient-airtemperature and ambient-air humidity. The only significant variable is therefore soil temperatureand it is worthy discussing those results by considering the effect of temperature on homeostaticcapacity of soil.

It is well known that soil system is complex and dynamic and it exhibites a good buffering effect,in the sense that it can "absorb", within certain limits, physico-chemical perturbations, withoutmodifing significantly its conditions. The complex interactions above mentioned also include thebehaviour of soil microbial-flora, which can influence, by its metabolism, physico-chemicalproperties of soil.

Particularly, PERA and FILIPPI (1987) and FILIPPI and PERA (1989) showed that the protectiveresponse to Fusarium oxysporum f. sp. diaruhi, subsequent to amending soil with sterile or nonsterile compost, is caused by the growing of soil zymogenic fraction.

In this case the metabolism of microbial groups has been enhanced by organic material atdisposal.

In the case studied in the present work, microbial population are not sensitive to temperaturevariations if no compost has been added. Therefore no changes occur in the interactions betweenpathogen, microbial groups and root. On the contrary when compost has been added, density anddistribution of microbial populations significantly change when temperature changes and the abovementioned interaction significantly change, causing a protective effect. It is in this broad sense thatmicrobial populations show an antagonist action toward the pathogen (antibiotic release, competi­tion with respect to food and growing places etc.).

A further and important observation is that plants exhibited, if protected, a suffering vegetativelook, as if they were suffering from a nutritional stress.

Such a case might be due to a less disposal of nutrients near the roots, caused by increasedmicrobial population, which need of great quantity of nutrients.

This observation is congruent with results reported in Figs. 2, 3,4, in which it can be seen thatmost evident protections of plants, characterized by the presence of a stressed vegetative look, occurwhen microbial populations exibit greatest increases.

Exists, furthermore, the suspicion that the stress state of the plant might inhibit the pathogenicactivity of Fusarium oxysporum f. sp. dianthi by some mechanism which has to be investigated.

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Authors' address: Dr. C. FILIPPI and Dr. A. PERA, Istituto di Microbiologia Agraria dcll'Universita di Pisa, Centro diStudio per la Microbiologia del Suolo, CNR, Pisa, Via del Borghetto 80, 56100 Pisa, Italy.