Biol Fertil Soils (1991) 11:13-17 Biology and Fertility o f S o i ~ s

�9 Springer-Verlag 1991

Effect of phosphonate on the rhizosphere microflora and the development of root rot (Phytophthora cinnamomi) in avocado (Persea americana) and pepper-corn (Schinus molle) tree seedlings

P. Wongwathanarat* and K. Sivasithamparam Soil Science and Plant Nutrition, School of Agriculture, The University of Western Australia, Nedlands WA 6009, Australia

Received February 27, 1990

Summary. Application to soil of 1 g (recommended rate) or 10 g 1-1 of phosphonate did not affect the numbers of bacteria and fungi nor the proportions of actinomycetes and fungi antagonistic to Phytophthora cinnamomi. Foli- ar phosphonate applications to avocado seedlings (Persea americana) did not affect microbial numbers or the pro- portions of microbes in the rhizosphere capable of antag- onizing P. cinnamomL Mycelium of P. cinnamomi and zoospores of R palmivora did not appear to respond to diffusates from excised roots of phosphonate-treated avo- cado and pepper-corn tree (Schinus molle) seedlings, re- spectively. However, less extensive lesions were observed on the roots of fungicide-treated avocado and pep- per-corn tree seedlings exposed to R cinnamomi and R palmivora, respectively. The reduction in R cinnamomi infection on pepper-corn tree seedlings appears to be brought about by additive rather than interactive effects of the resident soil microflora and foliar-applied phos- phonate.

Key words: Phosphonate - Soil microorganisms - Rhi- zosphere - Phytophthora cinnamomi - Phytophthora palmivora - Root rot - Avocado - Schinus molle

Soil microorganisms have an important role in control- ling Phytophthora root diseases (Broadbent et al. 1971; Broadbent and Baker 1974a, b). Suppression of root rot in avocado groves at Mt. Tamborine, Queensland, in Aus- tralia is a good example of the role of soil microorgan- isms in the natural suppression of disease incidence or se- verity, although this suppression is probably not due to soil microorganisms alone. The major group of soil mi- croorganisms that have an important role in suppressing the activities of soil-borne plant pathogens (e.g.P. tin-

* Present address: Chumporn Horticultural Research Centre, Sawi, Chumporn, Thailand

Offprint requests to." K. Sivasithamparam

namomi) are actinomycetes, bacteria, and fungi (Cook and Baker 1983). Interactions between soil microorgan- isms and soil-borne plant pathogens occur not only in the soil, but also in the rhizosphere. These interactions may be influenced by chemical substances (exudates) released by living roots (Curl 1982).

The effect of the interactions between soil microor- ganisms and soil-borne plant pathogens at the soil/root interface on disease incidence or severity might be altered by the application of a fungicide to the soil or onto the plant leaves. The fungicide might act through the soil mi- croorganisms or the host plant. Some reports have indi- cated that the application of certain fungicides caused changes in the composition and numbers of soil microor- ganisms which, in turn, affected disease incidence or se- verity. For example, changes in the composition of soil microbial populations brought about by the application of pentachloronitrobenzene have been shown to affect the severity of the damping-off disease caused by Pythi- um (Gibson et al. 1961).

Phosphonate is a new systemic fungicide which is used as a foliar spray or a trunk injection to control avo- cado root rot. It is very effective in controlling the disease in vivo (Pegg et al. 1985) although it has a low fungicidal activity in vitro. Several theories have been formulated to explain the mode of action of this successful fungicide following examination of its activity on Phytophthora spp. within the host tissues (Cohen and Coffey 1986). There is no information, however, on the potential action of phosphonate on the rhizosphere microorganisms, through root exudates from treated plants.

The aim of the present study was to investigate the ef- fect of phosphonate on P. cinnamomi infection in avoca- do or pepper-corn tree seedlings and to determine wheth- er any disease-reduction effect associated with the appli- cation of the fungicide is mediated through changes in the bulk soil microflora and/or rhizosphere microorgan- isms. An important part of this study involved monitor- ing changes in the proportions of microorganisms capa- ble of producing antibiotics as a mechanism of antago- nism (Baker and Cook 1974). P. palmivora was used in-

14

s t ead o f P cinnamomi w h e n s t u d y i n g z o o s p o r e s b e c a u s e

o f t he ease w i t h w h i c h t h e f o r m e r p r o d u c e d s p o r a n g i a

a n d r e l ea sed z o o s p o r e s in p u r e cu l tures . P e p p e r - c o r n t ree

s eed l ings were u s e d in p l ace o f a v o c a d o seed l ings in s tud -

ies w i t h an a sep t i ca l l y r a i sed h o s t b e c a u s e it was n o t p o s -

s ible to a sep t i ca l ly g e r m i n a t e a n d m a i n t a i n large n u m b e r

fo a v o c a d o seed l ings .

Materials and methods

Soil

counts. Plates were incubated at 22 ~ -+2 ~ in darkness for 4, 10, and 28 days in order to estimate the numbers of fungi, bacteria and ac- tinomycetes, respectively. To assess the frequency of occurrence of an- tagonistic microorganisms a minimum of four colonies were picked at random from each of the fifteen replicate plates used for the enumera- tion of bacteria, actinomycetes or fungi. Individual isolates were screen- ed for antagonism to P. cinnamomi on potato dextrose agar at 22~ ~ using the paired colony test. The proportion of isolates in each group that clearly inhibited the growth of the pathogen was com- puted by:

No. actinomyeete/bacterial/or fungal antagonists

Total no. actionmycete/bacterial/or fungal colonies isolated from enumeration plates

The soil used was collected from the top 15 cm of a field plot at the Uni- versity of Western Australia. The soil at this site is a Karakatta sand (McArthur and Bettenay 1960) which has been modified by the addition of organic matter and loam. After air-drying and sieving (2 ram), the soil showed the following properties: pH 6.2 (1 : 5 in water), 77.8 ppm P (NaHCO 3 extracted), 22 ppm NO3-N (calorimetric measurement, 1:5 in soil water), 7 ppm NH~--N (extracted in water and measured with a specific ion electrode after the addition of aluminium sulfate), 237.2 ppm K (NaHCO 3 extracted), 2.38% organic C, 384.2 ppm Fe (re- active) and 0.219 (dS m -1) conductivity.

The fungicide

Phosphorus acid was adjusted to pH 5.7-6.0 with potassium hydroxide to produce a solution which had an active ingredient of 200 g 1 -a phosphonate (HzPO3 -1 and HPO~2). In aqueous systems, phosphorus acid (H3PO 3) is in equilibrium with phosphonic acid (HzPHO3) so that essentially all of the molecules are in the phosphonic acid state. One way the two phosphonate anions (HPHO~ -1 and PHO32) can form is by the ionization of the phosphonic acid which has pKa values of 1.3 and 6.7 (Cohen and Coffey 1986). Most early reports used the terms phos- phorus acid and phosphite (Cohen and Coffey 1986) to denote the phosphonate moiety. According to the International Union of Pure and Applied Chemistry (1971), the correct term for the anionic form of phosphonic acid is phosphonate. This term is used in the present report. The fungicide used was a product of U. I. M. Agrochemicals (Aust.) Pty Ltd. Potassium phosphonate was diluted to prepare solutions of 10 g 1- l and 1 g 1 - 1 (recommended rate) phosphonate.

Pathogens

An isolate of P cinnamomi (A1 mating type), isolated from Banksia prionotes L. (Hardy and Sivasithamparam 1988) was used, with an iso- late of P palmivora (supplied by Dr. E. Davison) for the root diffusate test.

Host plant

The 4-month-old avocado (Persea americana Mills var. Guatemalan) and 2-week-old pepper-corn tree (Schinus molle L.) seedlings used as test plants were raised from seeds.

Effect o f phosphonate on microbial populations in soil

Soil samples (three 200-g dry soil aliquots) were treated with 60 ml water or a solution of phosphonate at 10g 1 -I (3rag g-1 soil) or 1 g 1-2 (0.3 mg g-a soil). A volume of phosphonate solution or water was ap- plied to bring the soil water content to 30% (weight/weight). Seventy grams of soil from each treatment were placed in a sterile 8-cm, diame- ter petri dish, and each dish was sealed with Parafilm | to minimize the moisture loss. The Petri dishes were incubated at 22 ~ 2 ~ in darkness for 21 days, and then three samples of soil (10 g each) were taken from each treatment, placed in 100 ml sterile water in plastic bottles, and shaken by hand 30 times. The resulting suspension was serially diluted in a 10-fold dilution series. Aliquots of the diluted suspension (0.1 ml) were spread onto Martin's (1950) medium for fungal counts and on to 1/5 M-32 agar (Ridge and Rovira 1971) for actinomycete and bacterial

Effect o f phosphonate on microbial populations in the rhizosphere o f avocado seedlings

Avocado seedling leaves (three seedlings per treatment) were sprayed un- til run-off with either water or a solution of phosphonate at 1 or i0 g 1-1 , using hand sprayers. The soil was covered with aluminium foil and paper towels to prevent any run-off reaching it. All seedlings were left for 7 days in a growth chamber at 22 ~ _+2 ~ and a 12-h light/darkness cycle. The light intensity above canopy was 243.46 ~tE s -1 m -2.

The roots (approximately 3 g) of each avocado seedling were careful- ly removed from the soil and were tapped gently to free them from large clods of soil. They were immersed in 100 ml sterile water in plastic bot- tles and shaken by hand 30 times. The rhizosphere microbial counts and calculation of the proportion of actinomycetes, bacteria and fungi an- tagonistic to P.. cinnamomi were done as described above.

Bioassay fo r phosphonate in root diffusates o f treated plants

Testing root diffusates on P. cinnamomi. Root diffusates following foli- ar application of phosphonate. Leaves of 4-month-old avocado seed- lings raised in plastic bags of 5 kg soil were sprayed until run-off with either water or a solution of phosphonate at 1 g 1-1. Seven days after spraying, the roots of three treated seedlings per treatment were recov- ered and washed free of soil, then cut into 100 pieces (=0.5 cm long), washed in sterilized water, surface-sterilized in 1 ~ NaOC1 for 2 min, washed again in sterilized water, blotted dry, and placed on seeded corn meal agar plates. The seeded plates were prepared by incorporating macerated mycelium of P. cinnamomi into cooled agar. The macerated mycelium was prepared by shaking mycelial mats within small McCart- ney bottles containing small glass beads and sterile water. Ten pieces of root were plated on each plate of corn meal agar: there were 10 plates per seedling and three replicate seedlings for each treatment.

Another 100 pieces of roots were cut from control avocado seed- lings. They were subjected to the procedure described above and dipped in 1 g 1-1 phosphonate solution to serve as a check.

The plates were incubated at 22~176 in darkness and examined daily. The inhibition of the fungus was rated on a scale divided into three levels (1, no inhibition; 2, moderate inhibition; and 3, strong inhi- bition).

Testing root diffusates on zoospores of P palmivora. The radicles of three aseptically raised pepper-corn tree seedlings were placed in a 5.0-cm diameter Petri dish. A 1.0-cm-diameter agar disk of R palmivora growing in cleared V-8 broth was rinsed with water twice and placed opposite the radicles in the same petri dish and 1.5 ml of cold sterilized water was added. Zoospores of R palmivora were released from zoosporangia into the water after 5-10 rain. Photographic re- cords were made of each radicle in the different treatments after the radicles had been exposed to the zoospores for 30-50 rain.

Effect o f phosphonate on the infectivity o f P. cinnamomi on avocado root

With minimal disturbance to the soil, the root tips of 4-month-old avo- cado seedlings, exposed by cutting small windows in the plastic contain-

er, were inoculated by plunging the root tip into a 0.5-cm-diameter V-8 agar plug colonized by P. c i n n a m o m i . Before the inoculation the seed- lings had been sprayed with either water or a solution of phosphonate at 1000 ppm and left for 7 days in a growth chamber as described above. Seven roots per seedling were each inoculated with a V-8 agar plug colo- nized by P. c i n n a m o m i while two roots were inoculated with an un- colonized V-8 agar plug as controls. Root lesions were assessed by mea- suring the extent of the discoloured tissue of the root 3, 5, and 7 days after inoculation. On the 8th day, each root was cut about 3 cm, above the margin of the discoloured tissues. The diseased roots were cut in- to~-0.7-cm long pieces and plated on P10 VP agar (Tsao and Ocana 1969) sequentially. The recovery of the fungus was then related to the position of the lesion on the root.

15

No. of root pieces yielding P. c i n n a m o m i

Total no. of root pieces plated

Root discolouration was rated on the following scale: 1, uninfected root; 2, roots slightly brown; 3, roots moderately brown; 4, roots dark brown; 5, roots dark brown and plant dead. The proportion of root pieces infected was the criterion used to assess the difference in disease severity between treatments.

Results

Effect of the interaction between soil microorganisms and phosphonate on the infection of pepper-corn seedlings by P. cinnamomi

Rhizosphere soil (approximately 3 g) was removed from the avocado seedling roots by gentle tapping and was incorporated into i00 ml nutri- ent solution (Agnihotri and Vaartaja 1967). The aliquots from these suspensions were then pooled to obtain 200 ml soil nutrient solution.

Seeds of the pepper-corn tree were pregerminated on layers of steril- ized tissue paper for 2 weeks and then transferred to sterile plastic con- tainers containing sterilized nutrient solutions until two leaves were formed. The seedlings were then divided into eight groups according to the treatment combinations listed below 1. Soil microorganisms (soil nutrient solution)+ P. c i n n a m o m i +

phosphonate 2. Soil microorganisms + P. c i n n a m o m i - phosphonate 3. Soil microorganisms - P. c i n n a m o m i + phosphonate 4. Soil microorganisms - P. c i n n a m o m i - phosphonate 5. No soil microorganisms (nutrient solution) + P. c i n n a m o m i +

phosphonate 6. No soil microorganisms + P. c i n n a m o m i - phosphonate 7. No soil microorganisms - P. c i n n a m o m i + phosphonate 8. No soil microorganisms - P. c i n n a m o m i - phosphonate.

The average pH of the solution before incubation was 5.2, for all treatments.

Seedlings exposed to soil microorganisms were allowed to equilibrate in glass vials (2 cm in diameter and 6 cm high) containing 10 ml soil + nutrient solution for 2 days before inoculation, while seed- lings not exposed to soil microorganisms were transferred to glass vials containing the nutrient solution only. The phosphonate was applied by dipping the seedling leaves in I g 1-1 phosphonate solution (recom- mended rate) for 3 - 4 min, while leaves of the seedlings not exposed to phosphonate were dipped in water. The R c i n n a m o m i treatments were inoculated with six V-8 agar disks (0.5 cm in diameter) colonized by P. c i n n a m o m i , and the control treatments were given six V-8 agar disks without P. c i n n a m o m L

The extent of disease was assessed by three criteria: (1) the propor- tion of infected root pieces; (2) a root discolouration rating; and (3) root fresh weight.

The proportion of root pieces infected was assessed by cutting the roots into small pieces (~0.3 cm long) and plating them on Plo VP agar with 33 randomly selected root pieces. The proportion of infected roots was calculated by:

Effect of phosphonate on microbial populations in soil

The application of the fungicide caused some changes to the properties of the soil (Table 1). There were sharp in- creases in soil K levels following the low (1 g 1-1) phos- phonate treatment, and of P, NHg--N and conductivity in the soil supplied with 10g1-1 of phosphonate. The numbers of bacteria and fungi, which ranged from 75 -- 80 X 106 g- 1 and 41 - 43 x 104 g- 1 soil, respectively, were not affected by the fungicide, but both concentra- tions of phosphonate led to drastic reductions in ac- tinomycete (Fig. 1) numbers. The proportions of ac- tinomycetes and fungi that were antagonistic to P. cin- namomi on potato dextrose agar ranged from 0.14 to 0.5 and 0.22 to 0.36, respectively, and were not affected by the phosphonate treatment at any of the concentrations tested. None of the isolates of soil bacteria that were screened showed any antagonism to P. cinnamomi

Effect of phosphonate on microbial populations in the rhizosphere of avocado seedlings

No significant difference in the rhizosphere numbers of actinomycetes, bacteria, and fungi was seen after the phosphonate treatment. Numbers (per g rhizosphere soil) ranged from 23.2 to 31.37<106, 3.87 to 6.41x106, and 8.86 to 11.28 x 104 for bacteria, actinomycetes and fungi, respectively. Similarly, no significant difference in the proportion of microflora antagonistic to R cinnamomi was evident.

Bioassay for phosphonate in root diffusates of treated plants

Testing of avocado root diffusates on the mycelium of P. cinnamomi. Sections of root from seedlings treated with the fungicide failed to inhibit P. cinnamomi on the seeded agar (Table2) although the root segments dipped in phosphonate did.

Table 1. Soil properties 21 days after treatment with fungicides and incubation at 22 ~ _+2 ~ in darkness

P NO~- -N NH~- -N K Organic Fe (ppm) (ppm) (ppm) (ppm) C (~ (ppm)

Conductivity (dS m - l)

pH (1 : 5 water)

Control (sterile water) 86.33 30.0 5.67 80.0 2.26 449 0.26 Phosphonate (1 g1-1) 96.0 30.0 4.33 219.67 2.16 467 0.31 Phosphonate (10 g 1 - i) 183.67 30.0 20.0 865.0 2.18 471.3 0.83

6.I 6.2

6.4

16

Table 2. The effect of avocado root diffusates on mycelial growth of Phytophthora cinnamomi at 1/5 strength corn meal agar

N u m b e r o f I n h i b i t i o n

p l a t e s t e s t e d r a t i n g

Control (water) 30 1 Phosphonate (1 g 1- ~) 30 1 Check (root pieces dipped in 10 2

1 g 1-1 phosphonate)

Inhibition ratings: 1, no inhibition; 2, moderate inhibition; 3, strong inhibition

6-

5

~ 4-

I.o

0 3-

X v ~ 2

r- _o 0 1 0

L.S.D..0t = 2.37

0 control 1.0 10.0

Phosphonate ( g . I -1)

Fig. 1. Effect of application to soil of two levels of phosphonate on counts of actinomycetes; L. S.D., least significant difference

12-

10"

E

r 8 -

e- 0 ' ~ 4-

n-

O o ~ 4 ~ ~, i'o

day

Fig. 2. The effect of foliar sprays with 1 g l- ~ phosphonate, 5 days be- fore inoculation with Phytophthora cinnamomi, on root lesion length; L. S.D., least significant difference

- - - - o - . - control -~ 1 g . I 1

Testing of root diffusates on infection of pepper-corn tree seedlings by zoospores of P. palmivora. There was no vi- sual difference among treatments in the responses of zoospores of R palmivora to root diffusates nor was there a pronounced accumulation of zoospores around the root surface of pepper-corn tree seedlings in any of the treatments. The distribution of zoospores on the plate was random.

Effect of phosphonate on the infectivity of P. cinnamomi on avocado root

The extent of root lesions on avocado seedlings was sig- nificantly decreased with increasing concentrations of phosphonate spray on the leaves of the seedlings (Fig. 2).

Interactive effect of soil microorganisms and phosphonate on the degree of infection of pepper-corn tree seedlings by P. cinnamomi

There was no significant interaction between soil micro- organisms and phosphonate that affected the infection of pepper-corn tree seedlings by P. cinnamomi (Table 3). Soil microorganisms were more effective in reducing the degree of root infection caused by P. cinnamomi than phosphonate (Table 3).

Discuss ion

The fungicidal effect of phosphonate does not appear to be mediated by microorganisms in the bulk soil or in the rhizosphere. The relative proportions of microbes antag- onistic to P. cinnamomi, either in the rhizosphere or in the bulk soil, were not affected by application of the fun- gicide. Although only the numbers of soil actinomycetes were reduced by the fungicidal drench, changes in the numbers of bacteria and actinomycetes were recorded at high rates of fungicidal application (P. Wongwathanarat and K. Sivasithamparam, unpublished data 1988) but not at the rate (1 g1-1) recommended for use on avocados. However, care needs to be taken in interpreting data from in vitro experimentations.

Investigation of the possible diffusion of phospho- nate or its derivatives from the roots of treated plants showed no detectable fungicidal or fungistatic activity, perhaps because there was no diffusion from the roots in- to the surrounding soil or because the assay had a limited sensitivity. The lack of chemotactic response by the zoo- spores of P. palmivora to the roots of pepper-corn tree seedlings is in contrast with the observations of Zentmyer (1980), who reported a positive chemotaxis by zoospores of P. cinnamomi on avocado roots.

The phosphonate was effective in reducing Phytoph- thora root rot on avocado in the present studies. The fun- gicidal effect of phosphonate on P. cinnamomi and on other Phytophthora spp. is widely accepted (Coffey and Bower 1984; Fenn and Coffey 1984). The persistence of phosphonate in avocado root tissues was confirmed by Ouimette and Coffey (1989) following foliar applications of potassium phosphonate. They showed that Phyto- phthora can actively accumulate phosphonate against a concentration gradient, raising the possibility that the level of phosphonate in the roots necessary to inhibit P. cinnamomi may be lower than that required if the mole- cule enters the fungus by passive diffusion. The mecha- nism(s) of fungicidal action of phosphonate observed in the present study appeared to be mediated through the plant, and confirmed the proposals of Bompeix et al. (1981), Guest (1984), and Saindrenen et al. (1988). In the absence of data on P levels in the leaves and roots it was

Table 3. Disease severity (mean of three values) in Phytophthora cinnamomi, and phosphonate)

pepper-corn seedlings due to interactions among three

17

major factors (soil microorganisms,

Treatment Proportion of root pieces Root discolouration Fresh root pH after 21 days' infected by P.c. index a (range 1 - 5 ) weight (g) incubation

Soil micro + P. cinnamomi + Phosphonate 0.12 Soil micro +P. cinnamomi- Phosphonate 0.21 Soil micro - P. cinnamomi + Phosphonate 0 Soil mic ro -P , cinnamomi- Phosphonate 0 No soil micro + P. cinnamomi + Phosphonate 0.43 No soil micro + P. cinnamomi- Phosphonate 0.56 No soil micro + P. cinnamomi- Phosphonate 0 No soil m ic ro -P , cinnamomi-Phosphonate 0

LSD (P< 0.05) 0.11 LSD (P< 0.01) 0.15

3.70 0.04 7.54 3.79 0.04 7.62 3.11 0.04 7.66 3.25 0.04 7.67 3.39 0.05 7.62 3.89 0.04 7.58 2.07 0.05 7.69 1.68 0.06 7.85

0.61 NS 0.80 NS

Soil micro, soil microorganisms; P.c., P. cinnamomi; LSD, least significant difference a Index: 1, uninfected root; 2 roots slightly brown; 3, roots moderately brown; 4, roots dark brown; 5 roots dark brown and plant dead

not possible to eliminate the possible P nutritional ef- fects. Ouimette and Coffey (1989), however, proposed that "phosphonate is not oxidised to phosphate by plant".

Even though the fungicide effect on the disease was not mediated through soil microorganisms, soil microor- ganisms were still able to play a significant role in reduc- ing the extent of the disease, suggesting that there may be some form of integrated control when fungicidal control of the root rot caused by P. cinnamomi is carried out in the field.

Acknowledgments. We thank Dr. A. L. J. Cole for help in the prepara- tion of the manuscript and Mr. P. McR. Wood for valuable advice on the fungicide. P.W. thanks the Australian Cooperation (Thai - World Bank) National Agriculture Research Project Scheme and the Govern- ment of Thailand for a studentship award.

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