Controlling of fusarium wilt in carnation with bark compost

Biological Wastes 22 (1987) 219-228

Controlling of Fusarium Wilt in Carnation with Bark Compost

A Pera Centro di Studio per la Microbiologia del Suolo, CNR Pisa, Via del Borghetto 80, 56100 Pisa, Italy

& C. Filippi Istituto di Microbiologia Agraria e Tecnica, Universitfi di Pisa, Via del Borghetto 80, 56100 Pisa, Italy (Received 10 February 1987; revised version received 6 March 1987; accepted 12 March 1987)

A BS TRA C T Poplar bark compost has been tested for its ability to control fusarium wilt caused by the phytopathogenic fungus Fusarium o x y s p o r u m f sp. dianthi on carnation plants. Compost was obtained through eomposting poplar bark previously inoculated with specific bacteria,following procedures to increase the antagonistic effect of such a substrate against Fusarium o x y s p o r u m f sp. dianthi. Poplar bark compost was added to a soil naturally heavily infested by Fusarium oxysporum f sp. dianthi (3000 units g - l ) and previously cultivated with carnations. Experimental tests were carried out in both greenhouse and open field. A visual inspection to evaluate mortality of carnation plants was performed ever)' 15 days. In greenhouse experiments protection against fusarium wilt was higher than 40% in compost-amended soil when compared to the control until the 60th day of the carnation cultural cycle. On the other hand, no significative difference wasfound among different treatments in open .field experiments. 219 Biological Wastes0269-7483/87/$03'50 © Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain

A. Pera, C. Filippi

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INTRODUCTION The use of compost derived from waste from sawmills (bark, sawdust, etc.) to control several phytopathologies has become more widespread of recent years in the cultivation of ornamental plants in containers (Daft et al., 1979; Hoitink, 1980; Spencer & Benson, 1981, 1982; Chef et al., 1983). Results with field crops are inconsistent. Larch-bark compost was used in the Nagano Valley, Japan, to control Fusarium brown rot in Chinese Yam (Sekiguchi, 1977) and this pathology was held at bay. The same compost inoculated with microorganisms was unable to control fusarium wilt in tomato (Fusarium oxysporum f. sp. lycopersici) in naturally infected soil (Kato et al., 1981): The use of ammoniated Douglas fir bark on soil infested with Phytophtora fragariae gave very good protection to strawberries in the first two years after treatment. Unfortunately, the disease returned as badly as ever in the fourth year both in amended and untreated soils (Vaughn et al., 1954). The ability of these composts to suppress phytopathologies depends on several factors such as type, level of maturity, and physical, chemical and biological properties, which are optimised by correct composting (Hoitink et al., 1977; Nelson & Hoitink, 1982; Nelson et al., 1983). This ability may also be enhanced by the addition of specific antagonist microorganisms (Fahy el al., 1981; Nelson & Hoitink, 1983). The aim of the present research was to test the antagonistic qualities of poplar bark compost towards Fusarium oxvsporum f. sp. dianthi in soil where the disease was edemic to carnation.

METHODS

Composting process The starting material was 4 tonnes of poplar bark, broken to a diameter of less than 2 cm, to which 4.8 kg urea and 2.4 kg perphosphate was added. The composition of the starting material is reported in Table 1. TABLE 1 C o m p o s i t i o n o f the Starting Material

Poplar bark, N + P o D M = Dry matter.

Moisture (%)

pH

58

7"6

Ash Organic C (% DM") (% DM") 21"02

49.7

Total N (% DM")

C/N

0"82

60.6

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Moisture content was corrected to roughly 58% by the addition of water. The resulting mixture was placed on a covered yard in trapeze-shaped windrows 1.7 m in height, 2.5 m in width and 4 m in length. The static pile method with forced ventilation from the bottom was used as described by Finstein (Finstein, 1980; Finstein et al., 1980a; 1980b). From the end of the thermophilic stage (roughly two months from the onset of composting) until its completion the windrows were turned once a month, during which operation 40 litres of 108 cells per millilitre of bacterial suspension of antagonists to Fusarium oxysporum f. sp. dianthi was added. Composting was carried out for six months and samples were taken periodically for analysis. Each sampling was made at four different depths (10, 60, 90, 120 cm) in eight different points. All data reported in this paper derive from the mean of two independent samplings.

Chemical and biological characteristics of compost Chemical analysis Dry weight was determined after drying at 105°C for 24 h. Organic matter was determined by combustion at 550°C. Humic matter was extracted by shaking a 5g sample in 100ml 0-1M NaOH in a nitrogen flow at room temperature for 24 h. Organic and humic C was determined by oxidation with Cr2 O2-. Total N was determined following Bremner's (1965) indication. Biological tests Phytotoxicity was tested in vitro on Lepidium sativum by the method of Zucconi et al. (1981a,b). Testing for substances inhibitory to Fusarium oxysporum was carried out on compost extract obtained by the method Zucconi et al. (1981a). The extract was passed through an F P U 30/3 Schleicher and Schull filter, 0.22 #m; 9 m m disks of absorbent paper (Oxoid) were immersed in the filtrate then placed on plates of semi-solid Waksman agar (0.7% agar) and kept at room temperature for 4-5min. The plates were then inoculated with approximately 106 conids ofFusarium oxysporum and incubated at 27°C for 24-48 h. The presence of inhibitors was evaluated by the inhibition halos on the plates. As control, tests were carried out using sterile H20 and a solution of cycloheximide (Sigma) (1/~g ml - 1). Isolation and preparation o f antagonist inoculum Antagonists were isolated from samples of bark taken before the onset of composting, using the method described by Filippi et al. (1984). The

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procedure was repeated once a month immediately prior to turning of the windrows. Strains were multiplied in shake cultures in Waksman broth. One-liter flasks with 880 ml medium were sterilised at 121°C for 20 min then inoculated with one antagonist and placed in shakers at 25 rpm and 28°C for 36 h. The biomass was collected by centrifugation at 3000 rpm and re-suspended in 40 litres of sterile water. The final density of the suspension was adjusted by spectrophotometry to 108 cells per millilitre. Greenhouse trials Soil naturally infested with Fusarium oxysporum f. sp, dianthi (3000 units per gram) was obtained from a nursery in Sanremo (Italian Riviera) where the disease has caused bad damage to carnations. The soil was amended with 15% (w/w) 6-months-old poplar bark compost. The mixture was placed in a 4 x 1 × 0.2 m bench box, planted with 200 rooted carnation cuttings of the cultivar 'Chimera rosa'. In another bench box of the same dimensions with unamended soil 200 carnations were planted as control. The plants were observed every 15 days to determine the percentage mortality rate. Several infected plants were unprooted and cultures made from the tissues to determine the nature of the pathogen. 1-2 cm sections were made at the base of the stem and disinfected for 5 min with sodium hypochlorite, then placed on Potato Dextrose Agar (PDA) (Difco) in petri dishes and incubated at 27°C for 72-96 h using Dimock's (1948) indications. Field tests Tests were carried out at Sanremo during the period from July to October 1986 at the same nursery from which soil had been taken for greenhouse trials. Compost used was identical to that for the greenhouse trials. The ground on which trials were carried out (30 × 50 m) was divided as follows: one-third of the area was amended with 15% compost (w/w of 20cm of topsoil), one-third was amended with 30% compost (w/w), one-third was left untreated as control. Each portion was planted with 1300 'Chimera rosa' carnation plants. The three plots were randomised since the disease was present in different infestation intensities in this field. The same observations as for the greenhouse experiments were carried out every 15 days.

Bark as control o f a plant pathogen

223

RESULTS Physical, chemical and biological characteristics of the compost The matter resulting from 6 months composting possessed all the chemical and physical properties necessary for agronomic use. The parameters are summarised in Table 2. The compost showed no phytotoxicity towards crops which might be cultivated in its presence (Fig. 1); no substances inhibiting the development of Fusarium oxysporum were found, either. The number of microorganisms antagonistic to Fusarium oxysporum f. sp. dianthi isolated from fresh bark was low (15-20 CFU g-1) and from only two genera, Bacillus sp. and Pseudomonas sp. (classification carried out in our laboratory using Bergey's Manual (Bergey, 1974)). Of those isolated, three strains (two Bacillus sp. and one Pseudomonas sp.) were chosen to reproduce biomass for periodic inoculation of the compost since these showed the best inhibition towards the pathogen in plate tests (Filippi et al., 1984). Monthly checks on the compost to determine the colonising capacity of the antagonists inoculated showed that their numbers remained lower (103-10 `*g-~ dry weight) than the quantities of inoculum introduced.

Greenhouse trials Results of trials on carnations show that compost-amended soil gave plants almost total protection from this disease for the first 60 days of trials when TABLE 2

Changes in General Analytical Characteristics During the Composting Process Composting time (days)

Characteristics 0 pH Moisture (%) Ash (% DM) Organic matter (% DM) Organic carbon (% DM) Humified carbon (% DM) Humified carbon (% organic C) N total (% DM) C/N

30

60

120

180

7"6 58 21.02 78.98 49.70 5.67

7"8 56 24.33 75.67 41-10 4.77

8"1 49 27.07 72"93 32.40 5"06

8"2 41 30"92 69"08 27-80 6-79

8"2 38 40'50 59'50 2590 7.30

ll.4 0.82 60.6

11.6 0.84 48.9

15"6 0"98 33" 1

24"4 1.14 24"4

28.2 1"15 22"5

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OL

~,

I

I

o

I

60

I

I

I

12o

1BO

tm (J~) Fig. 1.

Evaluation of germination index in Lepidium sativum with aqueous bark compost extract.

compared to the controls; the latter presented a mortality rate of about 55% at the same date. During the following days differences in values in the two mortality curves tended to diminish until they became irrelevant at 120 days (Fig. 2).

Field trials Results from field trials on carnation cultivation in soil naturally infested with Fusarium oxysporum f. sp. dianthi showed a mortality rate almost the same in all the plots, whether amended with compost or controls. The trends 10o 0 ~ 0

------0

.J'

611

1

I

I 30

I

I 60

I

I 90

I

I

120 tlme(~s)

Fig. 2. Pattern of fusariosis infection in carnation cultivated in greenhouse. Direct observation of percentage mortality. O - - O , plants dead in control soil; 0 - - 0 , plants dead in compost-amended soil.

225

Bark as control ~?f a plant pathogen

I lOO

I

8C

~ so IlL .

_-

I

i

I

I

T

,

40

11

20

0

I

I tkN (dill)

Fig. 3. Pattern of fusariosis infection in carnation cultivated in open fields. Direct observation of percentage mortality. 0 - - 0 , plants dead in control soil; , - - i n , plants dead in compost-amended soil (15% w/w); A - - A , plants dead in compost-amended soil (30% w/w). Bars represent least significant difference for P = 0-05.

in mortality for the various periods of the carnation cultivation cycle are reported in Fig. 3. A slight difference was noted between the death rate in control plants and those with 15% compost compared to those with compost at 30% in the observations taken up to 60 days from planting. These differences, which do, in fact, disappear at 90 days, were found to be statistically irrelevant.

DISCUSSION Results obtained in controlling Fusarium oxysporum f. sp. dianthi disease by compost from poplar bark are not easy to interpret. They confirm other authors' findings of the difficulty of transferring controlled conditions in a greenhouse to field trials (Bowen, 1979, 1980; Baker & Cook, 1982). Although the soil ecosystem can be rigorously and scientifically reproduced, it is, in fact, difficult to control parameters such as moisture, temperature, aeration, etc. It must be supposed, however, that these are not the only factors responsible for the disagreement in protection resulting under the different cultivation conditions. Results obtained can be interpreted as follows. When added to the open field, an organic substrate shows antagonistic properties towards the

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pathogen as a function of the intensity to perturb homeostasis of the soil (Alexander, 1971). Data from our experiments suggest that the organic matter, when applied to greenhouse bench boxes, has a much more decided impact than when applied in the field in the same, or even in double, the quantity. The protection from root pathogens afforded by the use of compost is certainly, to a great extent, due to microbial antagonism. This antagonism is obtained both by introducing microbial biomass into the soil with the organic matter (Kuter et al., 1983; Nelson & Hoitink, 1983; Hoitink & Kuter, 1984), and by the development of a telluric zymogen microflora, stimulated by the supply of easily-assimilated substances contained in the organic substrate added to the soil (Dommergues & Mangenot, 1970; Florenzano, 1983). In both cases the microbial population increases and reduces the virulence of the pathogen in the soil through its antagonistic activities (production of antibiotics, competition for space and nourishment, physical and chemical variations in the soil). The number of microbial populations is, however, destined to return to initial levels as time passes as a consequence of the selfregulating mechanisms present in every soil (Cook & Baker, 1983). The duration and intensity of this microbial behaviour is responsible for the changes in degree of protection as time elapses.

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