eradication of plant pathogenic fungi in soil and nursery potting mixtures with a mobile continuous...

Plant and Soil 42, 591-600 (1975) Ms. 2506

ERADICATION OF PLANT PATHOGENIC FUNGI IN SOIL AND NURSERY POTTING MIXTURES

WITH A MOBILE CONTINUOUS SOIL PASTEURIZER*

b y D. J . M I T C H E L L and L. N. S H A W * *

SUMMARY

A mobile continuous soil pasteurizer was evaluated for the eradication of soil-borne plant pathogenic fungi by assaying soil or infested potting mixture after treatment by selective cultural plating techniques and by host infection.

Populations of Pythium spp., Rhizoclonia solani, Fusarium spp., and other plant pathogenic fungi were eliminated from field soil after exposure to 80 3-2°C for 30 min.

Phytophthora parasitica, Pythium myriotylum, and Rhizoctonia solani were eliminated from an infested potting mixture treated at 60, 70, or 80°C. Aspergillus niger was recovered from the infested potting mixture at all of the temperatures tested. No papaya, peanut, rye, or tomato seedlings survived in potting mixture treated at a temperature below 40°C, but all seedlings survived in potting mixture exposed to 60, 70, or 80°C.

N o symptoms of phytotoxicity were noted in any of the plants at any of the treatments employed in this study.

INTRODUCTION

R o o t ro ts a n d seedl ing diseases caused b y soi l -borne fungi such

as species of Pythium, Phytophthora a n d Rhizoctonia m a y r educe

p r o d u c t i o n s ign i f i can t ly in n u r s e r y p l a n t beds 2 4 5 6 8 Whi le c h e m -

ical t r e a t m e n t s appl ied as sprays , drenches , or f u m i g a t i o n m a y p ro-

v ide a d e q u a t e con t ro l for these diseases, t h e y some t imes h a v e d raw- b a c k s such as p h y t o t o x i c i t y 2 6, t o x i c i t y to the user 8, a nd h igh ex-

pense, especia l ly if per iodic app l ica t ions are required . I n add i t ion , if

so i lborne p l a n t p a t h o g e n i c fungi are no t c o m p l e t e l y e r r a d i ca t e d in

* Florida Agricultural Experiment Station Journal Series No. 53,95. ** Assistant Professors of Plant Pathology and Agricultural Engineering, respectfully,

University of Florida, Gainesville, 3261 I.

592 D. J. MITCHELL AND L. N. SHAW

the plant beds, their populat ions m a y increase after plant material

considered to be clean is t ransferred to individual containers. This

si tuat ion m a y result in losses after extended periods of t ime ~

The principles and advantages of s team for the eradication of

plant pathogens from soil has been discussed thoroughly b y Ba-

k e r 2 3. To fully implement the advantages of s team t r ea tmen t

of soil, nursery operators in Florida have shown interest in equip-

ment tha t is mobile and can t reat large quanti t ies of soil or po t t ing

mixture under continuous operat ion wi thout destroying organic

matter .

The objective of this s tudy was to evaluate a mobile cont inuous

soil pasteurizer developed at the Univers i ty of Florida for the con-

trol of soil-borne plant pa thogens by assaying soil or infested po t t ing

mixture after t r ea tmen t by selective cultural plat ing techniques and

by host infection.

MATERIALS AND METHODS

The direct heat, continuous pasteurizer (Fig. 1) evaluated in this study is basically a heated, rotating, 10-foot drum through which the soil or potting mixture is conveyed as it is heated 12. The planting medium is fed into the upper end of the inclined drum by a variable-speed belt conveyor and water is introduced immediately before it enters the drum. The resulting planting medium-water slurry is heated by a liquefied petroleum burner directed into the lower or discharge end of the drum and is tumbled as it is conveyed slowly along the hexagonal sides of the rotating drum. The damp, heated planting medium is discharged into a wooden pallet box where it is held at the elevated temperature for the time necessary to eliminate pathogens. Thus, the temperature of the soil is regulated by the flow-rate of the planting material, the amount of water added to the material, retention time in the drum, torch setting, and the type of collection device.

For the experiment on the pasteurization of field soil, Arredondo fine sand was collected from a field planted in peanuts and known to contain high populations of several plant pathogenic fungi. The soil, which contained plant debris, was passed through the equipment at a rate (0.75 m3/h) that resulted ill a soil temperature of 80 ± 2°C and soil water content of 12% at the time of its emergence. After this temperature had been maintained for 30 min in the collection box, random samples were extracted from the container for assays.

The populations of Pythium spp. in untreated and pasteurized field soil were determined by preparing soil diluations of 1:20 and 1 : 100 in 0.3% water agar containing 300 ppm vancomycin hydrochloride and evenly spreading 1.0 ml on a solidified selective medium (PVPCNB) which consisted of corn meal agar (Difco) containing 5 ppm pimaricin, 300 ppm vancomycin hydrochloride,

MOBILE CONTINUOUS SOIL PASTEURIZER 593

Fig. 1. Mobile continuous soil pasteurizer consisting of a portable hydraulic power unit; variable-speed belt conveyor; regulated water source; varialole - speed, rotating, insulated drum ; a liquefied petroleum torch; and a collection

container.

and 100 ppm pentachloronitrobenzene 9. Each sample consisted of two dilutions and 10 plates per dilution. The dilute agar was washed from the surface of the selective medium after 36 hr of incubation in the dark at 30°C and the plates were observed for colonies of Pythium spp.

Levels of Rhizoctonia solani Kuhn in field soil were determined by evenly dispersing a 10-g air-dried sample in 10 ml of molten (45°C) 2,5% water agar containing 50 ppm streptomycin sulfate, cutting the solidified agar-soil mixture into 100 blocks, and placing 10 blocks on each of 10 petri dishes containing a selective medium (GDCS) developed by Ko and H o r a v. The plates were incubated for 36 hr at 30°C in darkness and observed microscopically for growth of R. sola~¢i.

Fusarium spp. were detected by preparing soil dilutions of 1:300 and 1 : 1,000 in 0.1% water agar containing 50 ppm streptomycin sulfate, and evenly spreading 1.0 ml of the dilution on a petri dish containing a solidified selec- t ive medium formulated by N a s h and S n y d e r 10. Ten plates, which were allowed to air dry for 5 days before use, were prepared for each dilution of each sample, and after incubation for 5 days at 30°C under fluorescent light, the plates were observed for colonies of Fusarium spp.

For the determination of populations of other fungi, 1.0-ml samples of soil dilutions of 1 : 1,000, 1:4,000, and 1:8,000 in sterile, distilled water were placed

594 D. J. MITCHELL AND L. N. SHAW

in each of 10 petri dishes per sample, and 15 ml of molten (45°C) potato dex- trose agar containing 0.1% Tergitol N P X (Union Carbide) and 50 ppm streptomycin sulfate (PDATS) were dispensed into each plate 14. The plates were incubated for 7 to 10 days at 25°C in light and observed for colonies of species of A spergillus, Cephalosporium, Cladosporium, Curvularia, Helminthosporium, Mycoleptodiscus, Penicillium, Rhizopus, Trichoderma, and other fungi.

For the host infection assay of the untreated or pasteurized field soil, ten 10-cm clay pots were seeded for each t reatment at the following rates: 5 corn (Zea mays L.), 3 peanut (Arachis hypogaea L.), 10 rye (SecaIe cereale L.) or 5 tomato (Lycopersicon esculentum Mill.) pregerminated, surface sterilized (0.5% sodium hypochlorite for 2 rain) seeds per pot. The pots were kept in a greenhouse at 25-32°C and soil moisture was maintained at field capacity or higher.

Two weeks after emergence, the seedlings were removed from the pots, roots were washed thoroughly and surface sterilized for 4 rain in 0.5% sodium hypochlorite, and root sections were placed on PDA containing 200 ppm streptomycin sulfate (PDAS) and on PVPCNB. The plates were observed after 2, 5, or 7 days of incubation at 30°C for fungi growing from the roots.

Inoculum for the infestation of pott ing mixture was obtained from cultures of Aspergillus niger van Tieghem and Pythium myriotylum Drechs. isolated from peanut, Phytophthora parasitica Dastur. isolated from Philodendron oxycardium Schott, and Rhizoctonia solani isolated from Scindapsus aureus Engler which were grown for 2 weeks at 25°C in l-liter erlenmeyer flasks containing 500 g of millet seed plus 300 ml of sterile water.

Approximately 500 g of inoculum of each fungus were mixed thoroughly in- to 50 kg of a potting mixture of coarse builder's sand and Florahome peat ( 1 : 1). The infested potting mixture was maintained in 15-cm clay pots in the greenhouse with daily watering for 2 weeks prior to the pasteurization experiments. After passage through the pasteurizer at approximately 1.1 ma/h, the pott ing mixture was collected and maintained for 30 min or more at temperatures of 25, 40, 60, 70, or 80°C.

Three 10-g samples of each potting mixture t reatment were dispersed in 10-ml aliquots of 2.5% water agar containing 50 ppm streptomycin sulfate. After the agar solidified, each sample was cut into 100 blocks which were evenly spaced on 10 petri dishes of PDAS, PVPCNB, or GDCS and incubated at 30°C in darkness. The PVPCNB and GDCS dishes were examined for P. myriotytum and P. parasitica, and R. solani, respectively, after 36 hr; PDAS dishes were examined after 5 days for A. niger and other fungi. Results were recorded as the percentage of blocks with each fungus.

Tile host infection assay of treated pott ing mixture was conducted as des- cribed for the soil host assay except that hosts were planted as two 2-week- old papaya (Carica papaya L.) seedlings, two unrooted Philodendron oxycardium cuttings, two pregerminated peanut seeds, 10 pregerminated rye seeds, or five pregerminated tomato seeds per pot. Ten pots of each host were planted for all treatments. Host roots were harvested and treated as in tile soil host assay for the detection of plant pathogens except that papaya and _P. oxycardium were allowed to grow for 4 weeks.

MOBILE CONTINUOUS SOIL P A S T E U R I Z E R 595

RESULTS

After t reatment in the continuous soil pasteurizer, potting mixtures and soil with an initial temperature of 80°C could be maintained in the collection container at 80~2°C for 2 h or more. Other temperatures employed in this s tudy were maintained in the collection containers for at least 30 min before samples of the planting medium were collected.

Important plant pathogens such as Fusarium spp., Pythium spp., and Rhizoctonia solani were eliminated and the populations of other

TABLE 1

Effect of t rea t ing field soil* a t 80°C with a continuous soil pasteurizer** on the incidence of fungi in field soil and in host roots

Incidence of fungus in soil or roots***

Soil Corn Peanut Rye Tomato Fungus Trea tment ppgt % % % %

Pythium spp. Unt rea ted 150 14 31 4 14 Pasteurized 0 0 0 0 0

Fusarium spp. Untrea ted 2,050 62 69 50 19 Pasteurized 0 0 0 7 5

Rhizoctonia solani Untrea ted 0.4 0 13 0 15 Pasteurized 0 0 0 0 0

Aspergillus niger Untreated 4,750 69 63 71 16 Pasteurized 1,300 69 75 7 85

Aspergillus spp. Unt rea ted 1,300 0 12 11 25 Pasteurized 700 0 12 0 0

Trichoderma spp. Untrea ted 1,450 100 75 58 4 Pasteurized 50 t 00 18 0 0

Rhizopus spp. Untrea ted 490 12 75 16 4 Pasteurized 100 15 50 4 70

Other fungi t t Untrea ted 16,500 50 69 100 62 Pasteurized 300 7 62 95 15

* Field soil was Arredondo fine sand from a peanut field known to contain various p lant pathogenic fungi.

** Soil temperature after emersion from the pasteurizer and for 30 rain thereafter was 80-b2°C.

*** Soil was assayed as soon as cool and roots were assayed 2 weeks after p lant emergence in test soil.

t ppg = propagules per gram of soil. I t Other fungi were species of Penicillium, Curvularia, Cladosporium, Helminthospo-

rium, Macrophomina, Selerotium, Cephalosporium, and Mycoleptodiseus. No impor tan t p lan t pathogenic fungi were found in pasteurized soil t reatments .

596 D. J . M I T C H E L L A N D L. N. S H A W

soil fungi were reduced in pasteurized field soil (Table 1). Other fungal pathogens such as Helminthosporiurn spp., Macrophomina phaseolina (Tassi) Gold, and Sclerotium rol/sii Sacc. were observed in low numbers in untreated soil but were eliminated in pasteurized soil. The host assay tests revealed no infections of host roots in pasteurized soil by Pythium spp. or R. solani. Fusarium spp. were isolated from 7 per cent of the rye plants and 5 per cent of the tomato plants.

T A B L E 2

Effect of t r e a t i n g in fes ted greenhouse p o t t i n g m i x t u r e * a t 80°C wi th a con t inuous soil pas teur ize r** on the inc idence of soi l -borne p l a n t p a t h o g e n s in p o t t i n g m i x t u r e and hos t

roo t s

% Inc idence of fungus in m i x or roots**

Philo- F u n g u s T r e a t m e n t t M i x t t P a p a y a P e a n u t dendron Rye

Aspergillus niger N I N P 43 30 50 80 62 N I P 9 20 30 60 21 I P 16 10 40 20 11 I N P 93 50 100 100 87

Pythium myriotylum N I N P 0 0 0 0 0 N I P 0 0 0 0 0 I P 0 0 0 0 0 I N P 18 20 34 40 63

Phytophthom parasitica N I N P 0 0 0 0 0 N I P 0 0 0 0 0 I P 0 0 0 0 0 I N P 6 20 0 50 0

Rhizoctonia solceni N I N P 0 0 0 0 0 N I P 0 0 0 0 0 I P 0 0 0 0 0 I N P 4 20 12 30 50

* The p o t t i n g m i x t u r e cons is ted of 50% coarse bu i lder ' s s and and 50% F l o r a h o m e pea t , and was in fes ted a t a r a t e of 500 g each of mi l l e t seed cu l tu res of A. niger, P. myriotylum, P. parasiiica a n d R. soIani per 50 kg of p o t t i n g m i x t u r e .

** T e m p e r a t u r e of the p o t t i n g m i x t u r e a f te r emers ion f rom the pas t eu r i ze r and for 30 ra in t he r ea f t e r was 80=t=2°C.

*** Soil was a s sayed as soon as cool and roo ts were a s sayed a f t e r 2 weeks of g rowth in

t e s t soil. t N I N P = no t infes ted, no t pa s t eu r i zed ; N I P = no t infested, p a s t e u r i z e d ; I P = in-

fes ted, pas t eu r i zed ; I N P = infested, no t pas teur ized . t t T e n - g r a m samples of cooled po t t i ng m i x t u r e were d iv ided in to 100 subsample s

which were p l a t e d on se lec t ive m e d i a ; d a t a recorded as % of subsample s w i t h g rowth of fungi .


Populations of the other predominant fungi were not eliminated in the soil and were isolated at various rates from the roots of host plants.

Of the four fungi added to the potting mixture, only A. niger was recovered from the mixture or from roots after pasteurization treatment at 80°C (Table 2). It was also isolated from the noninfested potting mixture.

No differences were observed in the appearances of hosts planted in uninfested, pasteurized potting mixture and in hosts planted in uninfested, untreated material. Thus, none of the treatments employed in this s tudy resulted in plants with symptoms of phytotoxicity.

In experiments on the treatment of potting mixtures at various temperatures, inoculum was increased to the rate of 2 kg of inoculum per 50 kg of potting mixture. At the termination of the test, all papaya, peanut, rye, or tomato seedlings planted in potting mixture treated at 60, 70, or 80°C remained alive (Table 3). No seedlings survived in potting mixture treated at 25 of 40°C. None of the P. oxycardium cuttings were killed at any of the temperature hea t - ments, but pathogenic fungi were isolated only from philodendron roots after growth in potting mixture treated at 25 or 40°C. Of the fungi added to soil, only A. niger was recovered from potting mixture or roots after exposure to 60°C or higher, but none of the host plants were killed.

DISCUSSION

The continuous soil pasteurizer employed in this s tudy retains the advantages of short time of exposure, low final temperatures, continuity of process, and low fuel cost of a flash-flame soil pasteurizer developed by N e w h a l l and S c h r o e d e r 11. Their equipment consisted of an inclined rotating drum with an oil burner directed into the discharge end, but no provision was made to add moisture to the soil during treatment. In the initial trials with the flash-flame type of pasteurizer at the University of Florida, the organic matter in high organic nursery mixtures was seriously degraded when the material was introduced into the machine at a typical seed bed moisture level (approximately 25 per cent H20). This experience prompted the introduction of a regulated amount of water into the

598 D. J . MITCHELL AND L. N. SHAW

TABLE 3

Effect of t r ea tment of infested pot t ing mixture* at various temperatures with a continuous soil pasteurizer on the incidence of soil-borne p lant pathogens in pot t ing mixture and in

host roots

% Incidence of fungus in mix or roots*** Temper- atures** Philo-

Fungus C Mixt Papaya Peanut dendron Rye Tomato

A. niger 25 t00 100 100 100 100 100 40 100 100 100 100 100 100 60 100 0 20 80 100 83 70 21 0 20 60 50 25

80 18 0 0 40 40 10

P. myHotylum 25 22 60 100 100 100 100 40 28 60 40 60 I00 I00

60 0 0 0 0 0 0

70 0 0 0 0 0 0

80 0 0 0 0 0

P. parasitica 25 14 70 0 60 0 0 40 6 30 0 0 0 0 6O 0 0 0 0 0 0

70 0 0 0 0 0 0 80 0 0 0 0 0 0

R. solani 25 12 60 60 80 25 55 40 3 40 10 20 16 33 60 0 0 0 0 0 0 70 0 0 0 0 0 0 80 0 0 0 0 0 0

* The pot t ing mixture consisted of 50% coarse builder 's sand and 50% Florahome

peat, and was infested at a ra te of 2.0 kg each of millet seed cultures of A. niger, P. myriotylum, P. parasitice, and R. solani per 50 kg of pot t ing mixture.

** Temperatures of the t reated samples of pot t ing mixture after emersion from the pasteurizer and for 30 min thereafter were 25~=2, 40~=2, 604-2, 70±2, 80~2°C.

*** Ten gram samples of cooled pot t ing mixture were divided into 100 subsamples which were plated on selective media; da ta recorded as percent of subsamples with growth of fungi.

* The pot t ing mixture was assayed as soon as cool and roots were assayed after 2 weeks of growth of peanut, rye, and tomato and after 4 weeks of growth of papaya and philodendron.


planting medium, and the degradation of the organic matter in the resulting slurry was eliminated.

While cool pockets or clumps in which plant pathogens, insects, or weed seeds might survive are problems with many steam pasteurization methods 1 2 a 11, the rotating drum in the continuous soil pasteurizer allowed even heat dispersal in the potting mixture and broke apart clumps.

All of the plant pathogenic fungi that might be of importance in the nursery industry were eliminated in the potting mixture or soil treated at 60, 70, or 80°C, but it should be noted that several fungi were recovered from soil or host roots after exposure to 80°C and that the potting mixture was never observed to be free of A. niger. Some predominantly saprophytic fungi such as A spergillus spp. and Pe- nicillium spp. are not eliminated at lower temperatures that nor- mally kill plant-pathogenic fungi 2, but they should have been de- stroyed at the high temperature of 80°C maintained for 30 min by the equipment used in this study. These fungi are common aerial contaminants and could have been easily reintroduced into the soil during transportation from the equipment site to the laboratory, in the laboratory, or in the greenhouse. Aspergillus niger was in- deed isolated from pasteurized as well as untreated potting mixture in high numbers even when it was not artificially infested. Since Fusarium spp. were isolated from a low percentage of roots of rye or tomato grown in pasteurized soil but not from the soil immediately after treatment, it is probably that this fungus was introduced by infested seed or contamination in the greenhouse.

Steam sterilization of soil at high temperatures may result in phytotoxici ty effects in some crops 18. S o n n e v e l d and V o o g t la recommended that soil be steam sterilized at 70°C instead of at 100°C to prevent undesirable side effects in lettuce, but that further research was required to show that control of the pathogenic soil organisms is adequate at the lower temperature. No symptoms of phytotoxici ty were observed on any of the crops at any of the temperature treatments employed in this study, and important plant pathogens were eliminated at 60°C or higher.

These studies demonstrate that this mobile equipment can be used in the rapid treatment of large quantities of soil or potting mixtures for the elimination of important soil-borne plant pathogenic fungi. The utilization of host assays of pasteurized material insured

600 MOBILE CONTINUOUS SOIL P A S T E U R I Z E R

that levels of pathogens in the soil too low to be detected by direct plating techniques did not escape elimination by pasteurization. Other work supports the conclusion that the temperatures attained with this equipment should also eliminate insects, nematodes and most weed seeds 3 11

Further investigations are being conducted to mechanize fully the equipment for directly filling various types of containers and maintaining desired temperatures therein for at least 30 min.

Received April 29, 1974

R E F E R E N C E S

1 A l d r i c h , R. A. and N e l s o n , P. E., Equ ipment for aerated s team t rea tment of small quanti t ies of soil and soil mixes. Plant Disease Reptr. 53, 784-788 (1969).

2 B a k e r , K. F. and R o i s t a e h e r , C. N., Heat t r ea tment of soil. pp. 123-137, I n K. F. B a k e r (Ed.) The U. C. sys tem for producing hea l thy container-grown plants. Cali- fornia Agr. Exp. Sta. Man. 23, 332 pp. (1972).

3 B a k e r , K. F. and R o i s t a c h e r , C. N., Principles of heat t r ea tment of soil. pp. 138-161, I n K. F. B a k e r (Ed.) The U. C. sys tem for producing heal thy container- grown plants. California Agr. Exp. Sta. Man. 23, 332 pp. (1972).

4 E n g e l h a r d , A. W., Mi l le r , H. N., D e N e v e , R. T., Etiology and chemotherapy of P y t h i u m root rot on chrysan themums . Plant Disease Reptr. 55, 851-855 (1971).

5 J a c k s o n , C. R., Control of P y th ium and Rhizoctonia in ch ry san themum plantings. Universi ty of Fla., Gulf Coast Exp. Sta. Mimeo Rept. 60-3 Nov. 16 (1959).

6 K n a u s s , J. F., Description and control of Py th ium root rot on two foliage plants species. Plant Disease Reptr. 56, 211-215 (1972).

7 Ko, W-H. and H o r a , F. K., A selective medium for the quant i ta t ive determinat ion of Rhizoctonia solani in soil. Phytopa thology 61, 707-710 (1971).

8 Mi l le r , H. N., Control of P y t h ium root rot of Chinese evergreen by soil fumigation. Proe. Fla. State Hort. Soe. 71, 416-419 (1953).

9 M i r c e t i e h , S. M., The role of Py th ium in feeder roots of diseased and symptomless peach trees and in orchard soils in peach tree decline. Phytopathology 61, 357-360 (1971).

10 N a s h , S. M. and S n y d e r , W. C., Quant i ta t ive est imations by plate counts of prop- agulus of the beau root rot Fusar ium in fields soils. Phytopathology 52, 567-572 (1962).

l l N e w h a l l , A. G. and S c h r o e d e r , W. T., New flash-flame soil pasteurizer. Cornell Univ. Agr. Exp. Sta. Bull. 875, 19 pp. (1951).

12 S h a w , L. N. and M i t c h e l l , D. J., A mobile continuous soil pasteurizer for organic pot t ing mixtures. A.S.A.E. Transact ions 18 (in press) 1975).

13 S o n n e v e l d , C. and V o o g t , S., The effects of soil sterilization with s team-air mixt- ttres on the development of some glasshouse crops. Plant and Soil 38,415-423 (1973).

14 S t e i n e r , G. W. and W a t s o n , R. D., Use of surfac tants in the soil dilution and plate count method. Phytopathology 55, 728-730 (1965).

eradication of plant pathogenic fungi in soil and nursery potting mixtures with a mobile continuous...

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