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Populations of Pseudomonas solanacearum biovar 3 in naturally infested soil
Soil Biol. Biochem. Vol. 16. No. I. pp. ST-61. 1984 Printed in Great Britain. All rights resemed
Cop)righr
0038-0717 51 S3.00 f0.00 % I9Y-I Pergamon Press Ltd
POPULATIONS OF PSEUDOMONAS SOLANACEARUM BIOVAR 3 IN NATURALLY INFESTED SOIL MELDA L. MOFFETTand B~BARA A. WOOD Plant Pathology Branch, Department of Primary Industries, Meiers Road. Indooroopilly. Queensland 4068 Australia (Accepred 20 July 1983)
Summary-Populations of Pseudomonassolunacearzrmbiovar 3 were monitored in a clay loam soil sampled from the root zone of infected tomato plants during 1978, 1979 and 1980. Soil numbers increased during symptom development and declined with the death of infected plants. The decline in population size in the soil was continuous where no cover crop was planted between the autumn and spring crops. This decline in population size was interrupted, however, following the planting of an oats cover crop numbers decreased with the ploughing under of the oats. Rainfall was associated with high soil numbers but soil temperature did not appear to directly affect population size. Soil populations in the root zone of susceptible tomato plants cultivar Floradel reached a maximum IOOO-foldgreater than in soil from the root zone of a resistant line. P. solanacearumsurvived in bare fallow soil for 21 months. Tomatoes planted 2 months later wilted rapidly.
30cm of soil. After planting but before the development of wilt symptoms, a further six samples, each comprising 6 bulked subsamples, were collected from the root zone of the susceptible cultivar Floradel. After symptom development, the locations of six wilted plants and six resistant plants were marked with permanent stakes, and composite soil samples collected from the root zone of both wilted and resistant plants. Every month from June 1978 until July 1980, 8-12 subsamples were collected from each of the six marked locations of the diseased plants. From December 1978 until August 1980 four samples each consisting of six subsamples, were taken every month from a bare fallow 3 x 0.5 m length row. Floradel tomato seedlings were planted in this area in October 1980. All samples were transported to the laboratory in an insulated container, held in a cold room at 4C and assayed for P. solanacearum within 24 h.
INTRODUCTION Pseudomonas solunacearum (Smith,
1896) Smith 1914
biovar 3 (Hayward, 1964) is the most important bacterial plant pathogen occurring in the coastal region of Queensland causing economic losses in tomato, tobacco, eggplant, capsicum and ornamental plants. The etiology of P. solanaceurum has been studied extensively (Smith, 1920; Kelman, 1953; Husain and Kelman, 1958; Kelman and Sequeira, 1965; Tanaka and Noda, 1973) since it was first recorded (Smith, 1896) but the factors affecting its survival in field soils have not been fully elucidated. Evidence for longevity of P. solanacearum in soil is largely indirect and in some instances based on glasshouse trials. This is partly because of the difficulties experienced in isolating P. solanacenrum directly from field soils (Karganilla and Buddenhagen, 1972; Tanaka and Noda, 1973; Nesmith and Jenkins, 1976). We report the monitoring of populations of P. solunncearum in a soil cropped with tomatoes for 3 yr and in a bare fallow soil.
Isolation of P. solanacearum
>l;\TERIALSAXD .METHODS Soil sampling
The field site studied was a clay loam soil at the Maroochy Horticultural Research Station, Nambour in southeastern Queensland and had a history of bacterial wilt. The soil has been described as a dark brown loam of crumb structure overlying red to red yellow clay subsoil with a percent content of organic carbon 2.5, coarse sand 19.0, fine sand 21.0, silt 22.0, clay 40.0, air dry moisture 4.1. The cropping history of the site during the sampling period is given in Table 1. Plant growth was maintained during dry periods with either overhead or trickle (after October 1979) irrigation and the bare fallow area was kept free of weeds. Before planting the first crop in February 1978, 12 soil samples, each comprising 10 bulked subsamples, were collected using a 15mm dia augur from the top 57
Each soil sample was thoroughly mixed and sieved (< 2 mm) if dry or mixed manually if wet. Two 20 g subsamples were taken, one dried overnight at 10X for dry weight determination, and the other added to 80 ml sterile distilled water and shaken vigorously for 30 min. A plant infection technique was used to isolate P. solanacearum. Tomato seedlings germinated on 1.5% water agar were transferred to slopes of Jensen’s medium (Vincent, 1970) supplemented with 0.5% KNO,, IO pg benomyl ml-’ and 1Opg metalaxyl ml-‘. They were maintained under artificial light for 8 days before inoculation and thereafter in a naturally-lit growth cabinet held at 28C. Duplicate inoculations were made by pipetting 1 ml of each soil dilution onto a tomato seedling and lacerating one or two roots with a scalpel. Confirmation of P. solunacearum in wilted seedlings and its presence in unwilted seedlings after 3 weeks, was determined by root isolation on sucrose peptone agar medium (Hayward, 1960) and incubating at 30’C for at least 72 h.
MELDA
L. MOFFETT and
BARBARY
h.
WOOD
Table I. Tix croppmg periods and the crops grown in a clay loam soil in LIhxh populations of Pseudomonas soiunaceurum biovar 3 m naturally infwsd ~011 were measured from 1975 co 1980 Ycu
Period
crop Fallow+ Tomato
?I June-?9 Sspt. 6 Oct.-l5 Jan. (1979) 7 Feb.-?0 June 20 June-?4 Oct. ?4 Oct.-9 Jan. (1980) 4 Mar.-2 July
Oats’ Tomaro Tomato Fallo\*t Tomato Tomato
Culur.3r or Line Floradel ~~u~c~ptible~ Floradel x VC9 F6 (resistant) Floradel Floradsl Floradsl Floradel
*Green manure crop. tArea maintained free of weds.
Estimation of soil population
The soil population of P. solanacearum was estimated by the dilution method (Fisher and Yates, 1963). As the lowest detectable number of P. solunacearum by the plant infection technique was 100 cells ml-’ (M. L. MoKett, unpublished Ph.D. thesis, University of Queensland), the number of P. solunacearum in a soil sample calculated by the method of Fisher and Yates was multiplied by a factor of 10’ to give the estimated soil population. Where P. solunacearum was not detected in a soil sample, a value 50% of the number of organisms found to be required to wilt one seedling was substituted for the zero value. calculated using Means were log .Ktransformation. Encironmental data
Daily rainfall and soil temperature data (at 1500 h and at a depth of IOcm) were recorded at a point 50m from the sampling site.
RESULTS P. solanacearum isolated from the experimental site was biovar 3 (Hayward, 1964).
F
The bacterium was not detected in soil samples until after wilt symptoms developed in February 1975 when the mean soil population was estimated to be 2.4 x IO’g-’ soil (Fig. I). The rapid increase in the soil population during March. reaching a mean maximum of 3.5 x IO6 organisms g-’ soil, corresponded to the progressive increase in bacterial wilt symptoms in the plants. It also corresponded to a period of vigorous vegetative growth and fruit set in healthy plants. Infected plants, although slightly stunted. had developing fruit on the first influorescence before their collapse. The peak in the size of the soil population (maximum detected. 2.8 x 10’ organisms gg’ soil) was followed by a sharp decline as the plants died during April and May. A population low occurred in June following the ploughing under of the remaining tomato plants. By this stage most of the visible tissue of the infected plants had disappeared. A slight rise in the population of P. sohnacearzrm was associated with the vegetative growth of the cover crop of oats. This was followed by a decline in soil numbers corresponding to the maturation and the ploughing under of the oats (Fig. 1). The population increased following the planting of the spring crop of tomato but the mean maximum population detected in December was loiver than that reached in
I
I
I
I
I
I
I
I
M
A
PA
J
JL
A
S
0
I
L
Tomato
N
0
I Oats
J
I Tomato
Fig. 1. Mean population size of Pseudomonassohnacearunt biovar 3 in soil from the root zone of naturally infected tomato plants cv. Floradel grown in a clay loam from February 1978 to January 1979. with rainfall and soil temperatures for the corresponding period.
Populations
J
I F
I M
I M
I A
of Pseudomonas solanacearum
I JL
I J
I A
in soil
59
I N
I 0
I S
1979 I
I 0
I
I
Fallow
Tomato
I J
Tomato
Fig. 2. Mean population size of Pseudomonas solanacearum biovar 3 in soil from the root zone of naturally infected tomato cv. Floradel plants grown in a clay loam from January 1979 until January 1980, with rainfall and soil temperatures for the corresponding period.
the autumn crop; the maximum soil number in any sample being 2.7 x IO’g-’ (Fig. 1). Increases in population size of P. solanacearum generally corresponded with high rainfall periods. Rain was recorded on six consecutive days in February and on every day in March and April except on two and three days, respectively. The prolonged wet period in November and December was associated with increasing numbers of P. solanacearum. Less rainfall at intermittent periods occurred from May to August. Soil temperature exceeded 20’C at time of infection, and during October and November, the period of lowest population size (Fig. 1). The lowest temperature recorded was 11°C. A similar population pattern was noted in 1979 (Fig. 2). The population size increased sharply with the onset of wilting, reaching a mean maximum in the autumn crop of 6.7 x 10’ g-’ soil. These soil numbers were lower than those recorded in 1978 as was the maximum number detected (2.1 x lo’ g-’ soil). The main deviation in 1979 was the gradual but continual decline in population. Because no cover crop was used between the autumn and spring crops, populations in this period could not be compared. The maximum number detected in the spring crop was 4.4 x 10’ g-’ soil and the mean maximum was lO’g_’ soil. Rainfall during 1979 was considerably less than in 1978. A recording of 244mm was made on one day in January and most other daily recordings, except in Table
2.
Mar. Mar. Apr. May
I
Tomoto
Fig. 3. Mean population size of Pseudomonas solanacearum biovar 3 in soil from the root zone of naturally infected tomato cv. Fforadel plants grown in a clay loam from January to June 1980, with rainfall and soil temperatures for the corresponding period.
February, were less than 20mm (Fig. 2). Soil temperatures were similar to those in 1978 (Figs 1 and 2). The pattern of population increase in 1980 following host maturity with wilt development was apparent, but this increase was not so marked as in the previous two autumn seasons. The maximum population figures were: mean maximum 3.3 x lo3 g-’ soil and maximum number detected 9.8 x lo3 g-’ soil. Rainfall in 1980 was greatest during wilt development. Soil temperatures did not vary greatly from 1978 and 1979 (Fig. 3).
The population of Pseudomonas solonocearwn biovar 3 in naturally infested soil from the root zones of a susceptible tomato cultivar and a rtsistant line
Date (1978) I3 31 I9 II
1980 L
Susceptible cultivar Resistant line (Floradel) (Floradel x VC9 F6) Max. population Mean Mean Max. population detected population population detected (cells g-’ soil) 2.4 2.0 3.5 6.8
x x x x
10’ IO’ IO” IO’
6x 2.8 x I.5 x I.7 x
IO’ IO’ IO6 IO’
3.5 x 1.8 x 4.7 x 1.7 x
102 IO’ IO’ IO’
7.9 x 3.5 x 2x 2.8 x
IO’ IO’ IO’ 10’
60
MELD-\
:
L.
SIOFFETT
and
BARBAM .A. WOOD
300
30
g
200
20
,1 ?!
E ; 0 ‘; E
0 IO
100
E”
i cE
?
0
:: $
,oJ
0
4: 0 m IO’ 97 Q.” IO2
~.-.\e_.---*-._*-*_e
.-.-.
‘;, tl 2 1
IO
OJFMAMJ
JL
1978
1979
A
S
0
N
D
J
F
M
A
M
J
JL
A
1980
Fig. 4. .Mean population size of Pseudotnonassolanacearumbiovar 3 in naturally infested bare fallow clay loam from December 1975 to August 1980 with rainfall and soil temperatures for the corresponding period.
Plants were young when infected in 1979 and 1980; being very stunted and smaller than those infected in 1978. Infected plants in 1979 and 1980 did not set fruit and collapsed earlier. A comparison was made of the population sizes in soil taken from the root zones of the susceptible tomato cultivar Floradel, and the resistant line, Floradel x VC9 F6. The population in soil in fhe root zone of susceptible plants increased for a longer period and was of a greater size than that in the root zone of resistant plants (Table 2). P. solunacearum was isolated from bare fallow soil from December 1978 until August 1980. There was an overall decline in population size during this period (Fig. 4). No relationship vvas evident between the decline in soil numbers and dry periods (Fig. 4). Isolation of P. solanacearum became increasingly difficult with time e.g. in 1980 P. sohnacearum was not recovered on four occasions. Seedlings planted in October 1980 commenced to wilt within 5 weeks of transplanting and half of the plants had wilted by 7 weeks. DlSCUSSlOh-
Population size of P. solunucearum biovar 3 in the soil increased in the presence of a susceptible host such as tomato. Soil numbers increased rapidly as wilting developed in the plant and declined sharply with its death. The number of P. solunuceurum cells in the soil during wilting is probably related to the rate of multiplication of the pathogen in the host and its release into the soil, both being greatest during symptom development. Soil moisture by facilitating exudation of the bacterium from diseased tissue affects soil population size. Kelman and Sequeira (1965) similarly found a rise and fall in population size as the plant wilted and died. The size of the soil population is also dependent upon the type of host, whether it is susceptible or resistant. A susceptible tomato cultivar such as Floradel has the potential of releasing at least lOOO-fold greater number of organisms into the soil than the resistant line Floradel x VC9 F6. Tanaka and Tomaru (1970) also detected fewer P. solanacearum cells
in soil around the roots of resistant than around those of susceptible plants. A steady decline in population size occurred in the soil following the death of the plant. The rate of decline in the absence of a host was continuous but where a non-host such as oats was planted, it was temporarily delayed (Fig. 1). This may indicate that oats provided a favourable site for survival and served as a short-term “sheltered” site (Graham and Lloyd, 1978; Graham, 1979). Such a possibility should be investigated further. The decline in population size ceased when a susceptible host was planted. Rainfall and thus soil moisture had some effect on changes in size of the soil population. High soil moisture has been reported to favour a rapid initial infection (Kelman, 1953) and increase in the rate of multiplication within the host (Gallegly and Walker, 1949). Soil moisture appears to be an important factor either directly or indirectly in determining the amount of inoculum returned to the soil from infected plants. Soil temperature did not appear to directly affect soil population size. In the absence of host plants, soil numbers declined with decreasing temperatures and despite a temperature rise, numbers continued to decline until infection of the spring crop. This evidence indicates that any effect temperature had on soil population size was possibly an indirect one by influencing disease development and thus the number of organisms exuded into the soil. Earlier work by Vaughan (1944) and Gallegly and Walker (1949) showed that soil temperature affected infection and symptom expression. The survival of P. solunucearrtm in bare fallow soil for up to 20 months confirmed earlier reports. Survival of P. solunacearrrm in bare fallow soil is obviously related to the strain. Thus the tobacco strain (race 1) appears to survive in soil for many years (Smith, 1944): the potato strain (race 3) from 2 to 3 yr (French, 1978; Lloyd. 1978); the banana strain for less than 2 yr (Sequeira. 1962). The long-term survival of the tomato strain appeared to be dependent upon small populations existing in an inactive state for extended periods.
Populations of Pseudomonas solanacearum in soil The pathogen was not isolated from all samples collected due probably to its non-random distribution. McCarter er al. (1969) also had difficulty in locating P. soiunacearum in known infested soils. Its non-random distribution probably reflects previous active sites where the pathogen had multiplied in susceptible hosts. Acknowledgements-We
thank Mr A. Diatoloff for his advice on the plant infection tests. Dr R. S. Allen and Mrs Janet E. Giles for statistical calculations. REFERENCES
Fisher R. A. and Yates F. (1963) Densities of organisms estimated by the dilution method. In Statistical Tablesfor Biological, Agricultural and Medical Research. 6th edn, pp. S-10. Oliver & Boyd. London. French E. R. (1978) Integrated control of bacterial wilt of potatoes. In Proceedings of the Second Regional Symposium on Potato Production-Southeast Asia and the Pacific. (R. V. Valdez. U. T. Rasco and L. J.
Harmsworth. Eds). International Potato Centre Far East and Southeast Asian Regional Office. Los Babes. Philippines. Gallegly &I. E. Jr and Walker J. C. (1949) Relation of environmental factors to bacterial wilt of tomato. Phytopathology 39, 936-946.
Graham J. (1979) Bacterial wilt of potatoes caused by Pseudomonas solanacearum E. F. Sm. The Journal of the Australian Institute of Agricultural Science 45, 123-124.
Graham J. and Lloyd A. B. (1978) Solanum cinereum R: Br.. a wild host of Pseudomonas solanacearum biotype I I. The Journal of the Australian Institute of Agricultural Science 44, 121126. Hayward A. C. (I 960) .4 method of characterizing Pseudomonas solanacearum. .Vature (London) 186, 401-406. Hayward A. C. (1964) Characteristics of Pseudomonas solanacearum. Journal of Applied Bacteriology 27, 265-277.
Husain A. and Kelman .4. (19%) Relation of slime production to mechanism of wilting and pathogenicity of Pseudomonas solanacearum. Phytopathology 48, 155-165. Karganilla A. D. and Buddenhagen I. W. (1972) Development of a selective medium for Pseudomonas solanacearum. Phytopathology 62, I3731 376. Kelman A. (I 953) The bacterial wilt caused by Pseudomonas solanacearum. A literature review and bibliography.
61
.Vorth Carolina .-lgricultural Experiment Station Technical Bulletin .Vo. 99.
Kelman A. and Sequeira L. (!964) Root-to-root
spread of
Pseudomonas solanacearum. Phytopathology 55, 304-309. Lloyd A. B. (1978) Survival of the potato strain of Pseudomonas solanacearum in soil. In Proceedings of the IVth International Conference on Plant Pathogenic Bacteria.
(Station de Pathologie vegetable et Phytobacteriologie. Ed.). August 27-September 3. Angers, pp. 873-878. McCarter S. M.. Dukes P. D. and Jaworski C. A. (1969) Vertical distribution of Pseudomonas solanacearum in several soils. Phytopathology 19, 1675-1677. Nesmith W. C. and Jenkins S. F. (1976) Selective medium for isolation of Pseudomonas solanacearum. In Planning Conference and Workshop on the Eco1og.v and Control of Bacterial Wilt caused by Pseudomonas solanacearum,
(L. Sequeira and A. Kelman. Eds). p. 121. North Carolina State University. Raleigh. Sequeira L. (1961) Control of bacterial wilt of bananas by crop rotation and fallowing. Tropical Agriculture (Trinidad) 39, 21 l-217.
Smith E. F. (1896) A bacterial disease of the tomato. eggplant, and Irish potato (Bacillus solanacearum n.sp.). United States Department of .-lgriculture Division of Vegetable Physiology and Pathology Bulletin 12. Smith E. F. (1920) The brown rot of Solanaceae. In An Introduction to Bacterial Diseases of Plants, pp. 177-201.
Saunders, Philadelphia. Smith T. E. (194-l) Control of bacterial wilt (Bacterium solanacearum) of tobacco as influenced by crop rotation and chemical treatment of the soil. United Stares Department of Agriculture Circular No. 692.
Tanaka Y. and Noda N. (1973) A study of factors governing the survival of tobacco wilt disease bacteria. Bulletin of the Okayama Tobacco Experiment Station .\-o. 32. pp. 81-93. Tanaka Y. and Tomaru K. (1970) Studies on the infection mechanism of Pseudomonas solanacearum E. F. Smith, the causal agent of bacterial wilt disease of tobacco. Bulletin of the Hatano Tobacco Experiment 68. pp. 67-86.
Station No.
Vaughan E. K. (1944) Bacterial wilt of tomato caused by Phytomonas solanacearum. Phytopathology 34, 443-458.
Vincent J. M. (1970) The assessment of nodulation and nitrogen fixation. In A Manual for the Practical Study of the Root-Nodule Bacteria. pp. 73-104. IBP Handbook No. 15, Blackwell, Oxford.
Cop)righr
0038-0717 51 S3.00 f0.00 % I9Y-I Pergamon Press Ltd
POPULATIONS OF PSEUDOMONAS SOLANACEARUM BIOVAR 3 IN NATURALLY INFESTED SOIL MELDA L. MOFFETTand B~BARA A. WOOD Plant Pathology Branch, Department of Primary Industries, Meiers Road. Indooroopilly. Queensland 4068 Australia (Accepred 20 July 1983)
Summary-Populations of Pseudomonassolunacearzrmbiovar 3 were monitored in a clay loam soil sampled from the root zone of infected tomato plants during 1978, 1979 and 1980. Soil numbers increased during symptom development and declined with the death of infected plants. The decline in population size in the soil was continuous where no cover crop was planted between the autumn and spring crops. This decline in population size was interrupted, however, following the planting of an oats cover crop numbers decreased with the ploughing under of the oats. Rainfall was associated with high soil numbers but soil temperature did not appear to directly affect population size. Soil populations in the root zone of susceptible tomato plants cultivar Floradel reached a maximum IOOO-foldgreater than in soil from the root zone of a resistant line. P. solanacearumsurvived in bare fallow soil for 21 months. Tomatoes planted 2 months later wilted rapidly.
30cm of soil. After planting but before the development of wilt symptoms, a further six samples, each comprising 6 bulked subsamples, were collected from the root zone of the susceptible cultivar Floradel. After symptom development, the locations of six wilted plants and six resistant plants were marked with permanent stakes, and composite soil samples collected from the root zone of both wilted and resistant plants. Every month from June 1978 until July 1980, 8-12 subsamples were collected from each of the six marked locations of the diseased plants. From December 1978 until August 1980 four samples each consisting of six subsamples, were taken every month from a bare fallow 3 x 0.5 m length row. Floradel tomato seedlings were planted in this area in October 1980. All samples were transported to the laboratory in an insulated container, held in a cold room at 4C and assayed for P. solanacearum within 24 h.
INTRODUCTION Pseudomonas solunacearum (Smith,
1896) Smith 1914
biovar 3 (Hayward, 1964) is the most important bacterial plant pathogen occurring in the coastal region of Queensland causing economic losses in tomato, tobacco, eggplant, capsicum and ornamental plants. The etiology of P. solanaceurum has been studied extensively (Smith, 1920; Kelman, 1953; Husain and Kelman, 1958; Kelman and Sequeira, 1965; Tanaka and Noda, 1973) since it was first recorded (Smith, 1896) but the factors affecting its survival in field soils have not been fully elucidated. Evidence for longevity of P. solanacearum in soil is largely indirect and in some instances based on glasshouse trials. This is partly because of the difficulties experienced in isolating P. solanacenrum directly from field soils (Karganilla and Buddenhagen, 1972; Tanaka and Noda, 1973; Nesmith and Jenkins, 1976). We report the monitoring of populations of P. solunncearum in a soil cropped with tomatoes for 3 yr and in a bare fallow soil.
Isolation of P. solanacearum
>l;\TERIALSAXD .METHODS Soil sampling
The field site studied was a clay loam soil at the Maroochy Horticultural Research Station, Nambour in southeastern Queensland and had a history of bacterial wilt. The soil has been described as a dark brown loam of crumb structure overlying red to red yellow clay subsoil with a percent content of organic carbon 2.5, coarse sand 19.0, fine sand 21.0, silt 22.0, clay 40.0, air dry moisture 4.1. The cropping history of the site during the sampling period is given in Table 1. Plant growth was maintained during dry periods with either overhead or trickle (after October 1979) irrigation and the bare fallow area was kept free of weeds. Before planting the first crop in February 1978, 12 soil samples, each comprising 10 bulked subsamples, were collected using a 15mm dia augur from the top 57
Each soil sample was thoroughly mixed and sieved (< 2 mm) if dry or mixed manually if wet. Two 20 g subsamples were taken, one dried overnight at 10X for dry weight determination, and the other added to 80 ml sterile distilled water and shaken vigorously for 30 min. A plant infection technique was used to isolate P. solanacearum. Tomato seedlings germinated on 1.5% water agar were transferred to slopes of Jensen’s medium (Vincent, 1970) supplemented with 0.5% KNO,, IO pg benomyl ml-’ and 1Opg metalaxyl ml-‘. They were maintained under artificial light for 8 days before inoculation and thereafter in a naturally-lit growth cabinet held at 28C. Duplicate inoculations were made by pipetting 1 ml of each soil dilution onto a tomato seedling and lacerating one or two roots with a scalpel. Confirmation of P. solunacearum in wilted seedlings and its presence in unwilted seedlings after 3 weeks, was determined by root isolation on sucrose peptone agar medium (Hayward, 1960) and incubating at 30’C for at least 72 h.
MELDA
L. MOFFETT and
BARBARY
h.
WOOD
Table I. Tix croppmg periods and the crops grown in a clay loam soil in LIhxh populations of Pseudomonas soiunaceurum biovar 3 m naturally infwsd ~011 were measured from 1975 co 1980 Ycu
Period
crop Fallow+ Tomato
?I June-?9 Sspt. 6 Oct.-l5 Jan. (1979) 7 Feb.-?0 June 20 June-?4 Oct. ?4 Oct.-9 Jan. (1980) 4 Mar.-2 July
Oats’ Tomaro Tomato Fallo\*t Tomato Tomato
Culur.3r or Line Floradel ~~u~c~ptible~ Floradel x VC9 F6 (resistant) Floradel Floradsl Floradsl Floradel
*Green manure crop. tArea maintained free of weds.
Estimation of soil population
The soil population of P. solanacearum was estimated by the dilution method (Fisher and Yates, 1963). As the lowest detectable number of P. solunacearum by the plant infection technique was 100 cells ml-’ (M. L. MoKett, unpublished Ph.D. thesis, University of Queensland), the number of P. solunacearum in a soil sample calculated by the method of Fisher and Yates was multiplied by a factor of 10’ to give the estimated soil population. Where P. solunacearum was not detected in a soil sample, a value 50% of the number of organisms found to be required to wilt one seedling was substituted for the zero value. calculated using Means were log .Ktransformation. Encironmental data
Daily rainfall and soil temperature data (at 1500 h and at a depth of IOcm) were recorded at a point 50m from the sampling site.
RESULTS P. solanacearum isolated from the experimental site was biovar 3 (Hayward, 1964).
F
The bacterium was not detected in soil samples until after wilt symptoms developed in February 1975 when the mean soil population was estimated to be 2.4 x IO’g-’ soil (Fig. I). The rapid increase in the soil population during March. reaching a mean maximum of 3.5 x IO6 organisms g-’ soil, corresponded to the progressive increase in bacterial wilt symptoms in the plants. It also corresponded to a period of vigorous vegetative growth and fruit set in healthy plants. Infected plants, although slightly stunted. had developing fruit on the first influorescence before their collapse. The peak in the size of the soil population (maximum detected. 2.8 x 10’ organisms gg’ soil) was followed by a sharp decline as the plants died during April and May. A population low occurred in June following the ploughing under of the remaining tomato plants. By this stage most of the visible tissue of the infected plants had disappeared. A slight rise in the population of P. sohnacearzrm was associated with the vegetative growth of the cover crop of oats. This was followed by a decline in soil numbers corresponding to the maturation and the ploughing under of the oats (Fig. 1). The population increased following the planting of the spring crop of tomato but the mean maximum population detected in December was loiver than that reached in
I
I
I
I
I
I
I
I
M
A
PA
J
JL
A
S
0
I
L
Tomato
N
0
I Oats
J
I Tomato
Fig. 1. Mean population size of Pseudomonassohnacearunt biovar 3 in soil from the root zone of naturally infected tomato plants cv. Floradel grown in a clay loam from February 1978 to January 1979. with rainfall and soil temperatures for the corresponding period.
Populations
J
I F
I M
I M
I A
of Pseudomonas solanacearum
I JL
I J
I A
in soil
59
I N
I 0
I S
1979 I
I 0
I
I
Fallow
Tomato
I J
Tomato
Fig. 2. Mean population size of Pseudomonas solanacearum biovar 3 in soil from the root zone of naturally infected tomato cv. Floradel plants grown in a clay loam from January 1979 until January 1980, with rainfall and soil temperatures for the corresponding period.
the autumn crop; the maximum soil number in any sample being 2.7 x IO’g-’ (Fig. 1). Increases in population size of P. solanacearum generally corresponded with high rainfall periods. Rain was recorded on six consecutive days in February and on every day in March and April except on two and three days, respectively. The prolonged wet period in November and December was associated with increasing numbers of P. solanacearum. Less rainfall at intermittent periods occurred from May to August. Soil temperature exceeded 20’C at time of infection, and during October and November, the period of lowest population size (Fig. 1). The lowest temperature recorded was 11°C. A similar population pattern was noted in 1979 (Fig. 2). The population size increased sharply with the onset of wilting, reaching a mean maximum in the autumn crop of 6.7 x 10’ g-’ soil. These soil numbers were lower than those recorded in 1978 as was the maximum number detected (2.1 x lo’ g-’ soil). The main deviation in 1979 was the gradual but continual decline in population. Because no cover crop was used between the autumn and spring crops, populations in this period could not be compared. The maximum number detected in the spring crop was 4.4 x 10’ g-’ soil and the mean maximum was lO’g_’ soil. Rainfall during 1979 was considerably less than in 1978. A recording of 244mm was made on one day in January and most other daily recordings, except in Table
2.
Mar. Mar. Apr. May
I
Tomoto
Fig. 3. Mean population size of Pseudomonas solanacearum biovar 3 in soil from the root zone of naturally infected tomato cv. Fforadel plants grown in a clay loam from January to June 1980, with rainfall and soil temperatures for the corresponding period.
February, were less than 20mm (Fig. 2). Soil temperatures were similar to those in 1978 (Figs 1 and 2). The pattern of population increase in 1980 following host maturity with wilt development was apparent, but this increase was not so marked as in the previous two autumn seasons. The maximum population figures were: mean maximum 3.3 x lo3 g-’ soil and maximum number detected 9.8 x lo3 g-’ soil. Rainfall in 1980 was greatest during wilt development. Soil temperatures did not vary greatly from 1978 and 1979 (Fig. 3).
The population of Pseudomonas solonocearwn biovar 3 in naturally infested soil from the root zones of a susceptible tomato cultivar and a rtsistant line
Date (1978) I3 31 I9 II
1980 L
Susceptible cultivar Resistant line (Floradel) (Floradel x VC9 F6) Max. population Mean Mean Max. population detected population population detected (cells g-’ soil) 2.4 2.0 3.5 6.8
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1980
Fig. 4. .Mean population size of Pseudotnonassolanacearumbiovar 3 in naturally infested bare fallow clay loam from December 1975 to August 1980 with rainfall and soil temperatures for the corresponding period.
Plants were young when infected in 1979 and 1980; being very stunted and smaller than those infected in 1978. Infected plants in 1979 and 1980 did not set fruit and collapsed earlier. A comparison was made of the population sizes in soil taken from the root zones of the susceptible tomato cultivar Floradel, and the resistant line, Floradel x VC9 F6. The population in soil in fhe root zone of susceptible plants increased for a longer period and was of a greater size than that in the root zone of resistant plants (Table 2). P. solunacearum was isolated from bare fallow soil from December 1978 until August 1980. There was an overall decline in population size during this period (Fig. 4). No relationship vvas evident between the decline in soil numbers and dry periods (Fig. 4). Isolation of P. solanacearum became increasingly difficult with time e.g. in 1980 P. sohnacearum was not recovered on four occasions. Seedlings planted in October 1980 commenced to wilt within 5 weeks of transplanting and half of the plants had wilted by 7 weeks. DlSCUSSlOh-
Population size of P. solunucearum biovar 3 in the soil increased in the presence of a susceptible host such as tomato. Soil numbers increased rapidly as wilting developed in the plant and declined sharply with its death. The number of P. solunuceurum cells in the soil during wilting is probably related to the rate of multiplication of the pathogen in the host and its release into the soil, both being greatest during symptom development. Soil moisture by facilitating exudation of the bacterium from diseased tissue affects soil population size. Kelman and Sequeira (1965) similarly found a rise and fall in population size as the plant wilted and died. The size of the soil population is also dependent upon the type of host, whether it is susceptible or resistant. A susceptible tomato cultivar such as Floradel has the potential of releasing at least lOOO-fold greater number of organisms into the soil than the resistant line Floradel x VC9 F6. Tanaka and Tomaru (1970) also detected fewer P. solanacearum cells
in soil around the roots of resistant than around those of susceptible plants. A steady decline in population size occurred in the soil following the death of the plant. The rate of decline in the absence of a host was continuous but where a non-host such as oats was planted, it was temporarily delayed (Fig. 1). This may indicate that oats provided a favourable site for survival and served as a short-term “sheltered” site (Graham and Lloyd, 1978; Graham, 1979). Such a possibility should be investigated further. The decline in population size ceased when a susceptible host was planted. Rainfall and thus soil moisture had some effect on changes in size of the soil population. High soil moisture has been reported to favour a rapid initial infection (Kelman, 1953) and increase in the rate of multiplication within the host (Gallegly and Walker, 1949). Soil moisture appears to be an important factor either directly or indirectly in determining the amount of inoculum returned to the soil from infected plants. Soil temperature did not appear to directly affect soil population size. In the absence of host plants, soil numbers declined with decreasing temperatures and despite a temperature rise, numbers continued to decline until infection of the spring crop. This evidence indicates that any effect temperature had on soil population size was possibly an indirect one by influencing disease development and thus the number of organisms exuded into the soil. Earlier work by Vaughan (1944) and Gallegly and Walker (1949) showed that soil temperature affected infection and symptom expression. The survival of P. solunucearrtm in bare fallow soil for up to 20 months confirmed earlier reports. Survival of P. solunacearrrm in bare fallow soil is obviously related to the strain. Thus the tobacco strain (race 1) appears to survive in soil for many years (Smith, 1944): the potato strain (race 3) from 2 to 3 yr (French, 1978; Lloyd. 1978); the banana strain for less than 2 yr (Sequeira. 1962). The long-term survival of the tomato strain appeared to be dependent upon small populations existing in an inactive state for extended periods.
Populations of Pseudomonas solanacearum in soil The pathogen was not isolated from all samples collected due probably to its non-random distribution. McCarter er al. (1969) also had difficulty in locating P. soiunacearum in known infested soils. Its non-random distribution probably reflects previous active sites where the pathogen had multiplied in susceptible hosts. Acknowledgements-We
thank Mr A. Diatoloff for his advice on the plant infection tests. Dr R. S. Allen and Mrs Janet E. Giles for statistical calculations. REFERENCES
Fisher R. A. and Yates F. (1963) Densities of organisms estimated by the dilution method. In Statistical Tablesfor Biological, Agricultural and Medical Research. 6th edn, pp. S-10. Oliver & Boyd. London. French E. R. (1978) Integrated control of bacterial wilt of potatoes. In Proceedings of the Second Regional Symposium on Potato Production-Southeast Asia and the Pacific. (R. V. Valdez. U. T. Rasco and L. J.
Harmsworth. Eds). International Potato Centre Far East and Southeast Asian Regional Office. Los Babes. Philippines. Gallegly &I. E. Jr and Walker J. C. (1949) Relation of environmental factors to bacterial wilt of tomato. Phytopathology 39, 936-946.
Graham J. (1979) Bacterial wilt of potatoes caused by Pseudomonas solanacearum E. F. Sm. The Journal of the Australian Institute of Agricultural Science 45, 123-124.
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spread of
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Saunders, Philadelphia. Smith T. E. (194-l) Control of bacterial wilt (Bacterium solanacearum) of tobacco as influenced by crop rotation and chemical treatment of the soil. United Stares Department of Agriculture Circular No. 692.
Tanaka Y. and Noda N. (1973) A study of factors governing the survival of tobacco wilt disease bacteria. Bulletin of the Okayama Tobacco Experiment Station .\-o. 32. pp. 81-93. Tanaka Y. and Tomaru K. (1970) Studies on the infection mechanism of Pseudomonas solanacearum E. F. Smith, the causal agent of bacterial wilt disease of tobacco. Bulletin of the Hatano Tobacco Experiment 68. pp. 67-86.
Station No.
Vaughan E. K. (1944) Bacterial wilt of tomato caused by Phytomonas solanacearum. Phytopathology 34, 443-458.
Vincent J. M. (1970) The assessment of nodulation and nitrogen fixation. In A Manual for the Practical Study of the Root-Nodule Bacteria. pp. 73-104. IBP Handbook No. 15, Blackwell, Oxford.