Behaviour of Phytophthora cinnamomi rands in different soils and water regimes

BEHAVIOUR OF PHYTOPHTHORA CINNAMOMI RANDS IN DIFFERENT SOILS AND WATER REGIMES ROSEMARY J. REEVES* Department

of Biological

Sciences. University

of Surrey. Guildford,

Surrey. GU2 5XH

(Accepted 21 Junr 1974) Summary-The effect of different soils, nutrient states and water regimes on the growth, sporulation and lysis of mycelial inocula of Phytophthoraciwzamomi has been examined. It has been observed that the requirements for chlamydospore and sporangium production in soils are relatively non-specific with respect to soil type. pH, percentage organic matter and the presence or absence of an additional food source. In contrast to chlamydospore production, however, production of sporangia in soil depends on a sufficiently low water suction pressure. In some soils a low percentage water content or a water content well below field capacity did not necessarily inhibit sporangium production. The pathogen was a good competitor for pieces of both fresh and rotting Castarzea satica radicles. Trichoderm uiride appeared to play a significant role in soil by lysing hyphae of P. cinnurnorni and inducing it to produce oospores. MATERIALS

INTRODUCTION Although

Phytophthom

cinnumotni

* Present address: London. SW7 2AZ.

Botany

Department.

Imperial

College,

Table

Soil

PH

1 2 3 4 5

5.3 7.3 5.0 4.5 5.0

AND METHODS

The activity of the fungus in five different soils was examined. Four of the soils were collected from various sites in Switzerland and one (soil 2) from a nursery in southern England, in another part of which P. cinrzur~~o~~~i is known to occur. A brief description of the soils is given in Table 1. A desorption curve for each soil was obtained by means of a pressure membrane apparatus (description in Rose. 1966), in the Soil Physics Department, E.F.A., Birmensdorf. Switzerland. The soils were stored moist in covered bunkers outside until required. They were air-dried in small amounts in shallow trays at room temperature l-2 days before required. An A? compatibility type P. cinmmorui isolate was obtained from a diseased Cu.stan~ sufiua seedling from a nursery in southern England and maintained on oatmeal agar slopes (oatmeal 30 g; agar 15 g distilled water to 1 litre). Air-dried soil was sieved through a 2 mm mesh, placed in 2OOg amounts in 850 ml glass pots and stored at 4°C for 24 h. The cooled, sieved soil was then held at -25°C for I-3 h. Crushed, distilled water ice was also cooled to -25°C and added to the cooled, sieved soils, in amounts calculated from the desorption curve of each soil to give the final pF value required, allowance being made each time for the initial moisture content of the air-dried soils. The ice/soil mixtures

Rands is an important soil-borne pathogen causing serious losses in a variety of crops, relatively little is known of its behaviour in soil. Zentmyer and Mircetich (1966) have shown that it is capable of surviving for several years in soil in the absence of a host. The exact mode of survival is not known, but is probably by resistant spores. The occurrence of chlamydospores of this fungus in soil has been reported by Hendrix and Kuhlman (1965). Mircetich and Zentmyer (1966) and M&din et a/. (1967) and oospores by Mircetich and Zentmyer (1966) and Reeves and Jackson (1972). Reports of the competitive saprophytic ability of this pathogen in soil have been conflicting. Kuhlman (1964) observed poor invasion of Douglas fir twigs by P. cinrmrnorni in non-sterile soil and no spread through this soil. Zentmyer and Mircetich (1966). however, concluded that this fungus had some competitive saprophytic ability, since it invaded wheat straw and dead avocado roots in non-sterile soil and grew up to 3 cm in non-sterile soil from a food base. This paper reports observations on the effect of different soils, nutrient states and water regimes on the behaviour of mycelial inocula of P. cinnamorni in soil.

I. Description

7” (w/w) Organic matter

of soils “/: Water (vol. H,O/w dried soil) at field capacity 41.5 60.6 55.7 25.4 23.0

2.45 2.95 I.4 0.67 0.89 19

Soil type and horizon Sandy loam Clay loam Podrol (A 1) Podzol (A2) Podzol (A2)

20

ROSHAKYJ. R~EVIS

were incubated at 4°C for 24 h and then at 22°C for 48 h bci’orc use. This method of moistening soil was adopted because it gave a relatively uniform final crumb structure, and uniform distribution of water, not obtained when water is sprayed on to the surface of soils or mixed into them. The microbial ~~opulation of soil I before and after air-drying and ilnmediately before use after moistening with ice was examined by dilution plate counts on a variety of agars. The results of these counts indicated that air-drying and remoistening in this way caused no major changes in the fungal, actinomycete or bacterial populations. The soils were also monitored for the presence of P/?~r~p/~~~~~ruspp. by inoculating apples (cultivar: Golden Delicious). No P~~~op~~f~o~~s were recovered. lnocula were prepared by growing P. cinnarnorni in sterile carrot juice (carrot I50 g; distilled water 1 litre) in Petri dishes for 48 h at 22°C. Small pieces of mycelium from these cultures were then transferred to fresh carrot juice and incubated for a further 4X h at 22°C. The Inyceiiun~ from these cultures was then broken up by rapid agitation with a straight sterile wire and pieces examined to check that no spores were present. The mycelial fragments were used to inoculate nylon mesh discs ( I&- IX mm dia and I mm mesh) which were then incubated for 36 h at 22‘C in sterile carrot juice. The discs and mycelium were thoroughly washed in 10 changes of sterile. distilled water and blotted dry with sterile, glass filter paper. Where a nutrient source was required, nonsterile 2 mm-thick slices (4 mm dia) of radicle or feeder root of C. sativn seedlings grown in Pcrlite [.lohns-Manville (Great Britain) Ltd.] were placed on the discs. Nylon mesh was used since preliminary experiments comparing growth, lysis and sporulation of the pathogcn when mounted on nylon mesh and in its absence in water and on moist soil surfaces, indicated that nylon mesh had little effect on these. The use of a liquid medium prevented damage of the hyphae before burial, such as occurs when the fungus is grown on, and then removed from. agar. The nylon discs bearing mycelium with or without a root slice were then buried to a depth of 2-3 cm in the moistened soils with as little disturbance to the soils’ structure as possible. The pots containing the soil and 14 I6 discs per pot were incubated at 22 + 1°C and weighed daily to give an indication of water loss

or gain thro~l,~hout the experiment, Similar pots containing 14-14 discs in 300 ml of distilled water were used as water controls. Five discs from each treatment and each soil were recovered daily and washed gently to remove adhering soil particles. Four of the discs were mounted in lactophenol and examined microscopically. Root pieces were gently crushed between two glass slides before being mounted in lactophenol blue and examined. One disc and, if present, root piece, were placed on P,,,VP agar (Ocana and Tsao, 1966) and incubated at room temperature to test for viability.

EXPERIMENTAL

(a) Effect ofa mrfrient source on hehmiour in soil The pF of the five soils was adjusted to ca. 2.6; the quantities of ice required for each 200g of soil are given in Table 2. The behaviour of myceliaf inocula of this pathogen on discs in the presence and absence of a food source and in root pieces in the five soils and a water control was observed over a period of 17 days. The results are summarized in Table 3 and below. Sporatgia. Sporangia were found in all soils and in the water controls. Direct and indirect germination of some sporangia occurred in all of the soils, but a proportion of unterminated sporangia persisted after mycelial lysis. To determine whether the delay in their production on the discs without a food source in soil 4 was due to lack ofwater, the rate of production of sporangia by mycelial inocula in suspensions (soil 5 g; distilled water 250 ml) of soil 4 and soil 1 was compared. Sporangia were observed on the mycelium in the soil 1 suspension after 24 h and in the soil 4 suspension after 8 days. Water was not, therefore, the limiting factor in soil 4. Chlamydospores. On discs chlamydospores were spherical, single or clustered and persisted after mycelial lysis. In root pieces, they were both inter- and intracellular. Those formed in cells were often very large, filling and assuming the shape of the cells. Others occurred two-four in a single cell and were spherical. Oospores. The stimulation of production of oospores in root pieces in soil by TtMod~~nn uiride has been reported and discussed elsewhere (Reeves and Jackson,

Table 2. Preparation and water characteristics of experimental soils

Soil

1 7 3 4 5

Ice (g/200 g air-dried soil) to give final pF ca. 2.6 74 100 42 12 16

Final ?j W 0 (ml tiTC$v dried soil) 369 50-O 21.0 5.9 8.0

Final % of field capacity 89 82.4 37.7 23.2 24.2

3

I + i

i /

i +

3

+Y

+

+i+

i++

+ + +++ +++

+ ++ +++

s3f

mr

0

3

t+z

z

aG13

+++

+-I-+

+++

+ +

f-w-

0

++

+ -t

++t

0

mr:

0

++-!+i+

mr

0

22

ROSEMARY

1972). They developed in root pieces in the three soils, i.e. I. 2 and 3. in which 7: ciride was abundant, but not in water or soils 4 and 5 from which this fungus was not isolated. Growtlz. Mycelium grew very quickly into all root pieces. completely colonizing them within 2-4 days. Hyphae from discs. with and without a food source, were observed growing up to 1 mm into pieces of organic matter in the soil around the discs. Chlamydospores could be observed in and sporangia on these pieces of organic matter. This invasive ability of P. cirmw~onzi was further demonstrated when pieces ofroot, previously buried in moist soil I for 6 days were placed on fresh, mycelial discs and buried. The fungus rapidly grew into and over these root pieces and persisted there until the end of the observation period (15 days) despite the presence of other soil microorganisms. LJY~S.In these experiments, the term lysis is used to include emptying of hyphae as well as cell wall disintegration. Extensive. although not complete lysis occurred in all of the soils within 17 days. However. in all but soil 2 lysis was not so rapid on the discs in the presence of a food source. By contrast. in soil 2 lysis was more rapid on discs in the presence of a food source. Intact and extensive Ph~mphthotw mycehum persisted in root pieces until they disintegrated despite the presence of large quantities of other fungal structures and bacteria. Rapid and extensive lysis on the discs was frequently associated with the presence of sporing 7: Gridr, the growth of which appeared to be stimulated in the vicinity of the Phyrophfhoru mycelium. Occasionally this fungus was seen coiling around the hyphae. This association between 7: uiridr and mycelial lysis of P. cirmumor~i in soil was also observed in the experiment described below (b). In soils 4 and 5 that did not contain detectable 7: riride, lysis was slower and was not particularly associated with any other microorganism. Confirmation of the ability of 7: viridr to cause the lysis of P. cimm~orni was obtained in further tests both in 11itro and in soil. When fresh discs of Plzytophflzora mvcelium were inoculated with 1.X x 10” and 1% x 10’ conidia of 7: viride (isolated from soil 3) and buried in soil 4 (from which 7: ciride could not be isolated) the rate and extent of mycelial lysis in this soil was greatly increased. III vitro experiments. involving the inoculation of 4-day-old carrot agar cultures of P. cimamorni with conidia of 7: riridr and treating Phyfoplzthora mycelium on carrot agar with cell-free filtrates of Eichoderma from 7-day-old cultures in modified Raulin Thom medium (Smith, 1968). further demonstrated the rapid lysis of P. cirmzrnomi mycelium by 7: viride.

The results of the experiment above indicated the presence of a food source had little effect on production of sporangia and chlamydospores in although some differences were observed in

that the soil, the

J. REEVFS

number and rate of production. It was, therefore. decided to test the effects of different water regimes on the production of these spores in soil. in the absence of an added nutrient source, although it was realized that a moisture/nutrient interaction might exist. Soil 2 was moistened with the appropriate amounts of ice to give the following water contents (:‘{, v/w): 50 (pF ca. 2.6): 30 (pF cu. 3.7): 20 (pF cu. 4.2); 15; 10 and 5. The pFs of the lower water regimes could not be measured since the apparatus used for measuring suction pressures was not suitable for pressures above 15 atm. The behaviour of mycelial inocula of P. cirlr~rrlonzi in this soil under different moisture conditions and in a water control was observed over a period of 26 days. Chlamydospores were produced within 5 days over the whole range of soil moisture regimes examined. although somewhat faster and in greater numbers in the moister soils. They persisted after mycelial lysis at all water levels. Sporangia and zoospores were observed only in the wettest soil (pF cu. 2.6) and in the water control. Extensive lysis took place at all water regimes, but was most rapid at 15 and 20 per cent. Generally less lysis occurred in the wettest and driest soils. However, extensive. if not complete. lysis occurred at all water regimes by the end of the experiments at 26 days. DISCUSSION

After death of its host. the mycelium of P. cimurnomi in the dead tissue will experience a decrease in available nutrients and increasing competition unless infection of a new host follows. Rapid colonization of organic matter including root pieces. with the formation of chlamydospores and sporangia, occurred in all soils in competition with the soil microflora. This suggests good, competitive saprophytic ability and supports Zentmyer and Mircetich’s (1966) conclusion that P. cinnumorni can be considered a soil-inhabiting fungus. Since extensive, although not always complete, lysis occurred in all of the soils tested within 17 days, mycelium of this fungus does not appear to be long-lived in soil outside root pieces or possibly other fresh organic matter. Survival of unlysed hyphae in the root discs at the end of the experiment, despite the presence of many other microorganisms, may have been due to some degree of protection from lytic organisms or a consequence of the higher prevailing nutrient levels. Observations made during these experiments and in aifro studies with 7: ciride indicated that this fungus may be a natural and vigorous antagonist of P. tinnumomi in soil. Weindling (1932) reported parasitism of PhJ~tophthoru purusiticu in soil by Trichodermu [although the identity of the fungi studied by Weindling is uncertain (Webster and Lomas, 1964)] and Lacey (1965) observed emptying of hyphae and sporangia and possible parasitism following contact between Phytophfhora in$,.sturn and 7: kide. More

Behaviour of Phvtophthora recently Dennis and Webster (1971) reported penetration of Phytophthora cactoruw and Phytophthora erythroseptica in vitro by two strains of T viride. The increase in the rate of mycelial lysis at 15 and 20 per cent moisture contents in the second experiment may have been due to more favourable conditions for the growth and development of antagonistic organisms such as 7: viride or to the Ph~tophthora mycelium becoming more resistant to lysis at the higher and lower moisture levels. Both of these suggestions are partly supported by Kouyeas’ (1964) studies in the moisture

relations

of soil fungi.

The persistence of chlamydospores and sporangia after mycelial lysis suggests that they may function at least as short-term survival units. Oospores probably play a similar role. but in these short experiments they were never observed in the root pieces in the absence of viable mycelium. The presence and survival of chlamydospores in soil have been described previously (Hendrix and Kuhlman, lY65;Zentmyer and Mircetich, 1966). Blackwell (1943) reported that sporangia of P. cacrortw ripen into conidia and survive for months under moderately dry conditions in vitro. Sporangia of some pythiaceous fungi are known to be capable of acting as survival units in soil (Zan, 1962; Lacey, 1965; Stanghellini and Hancock, 1971) and the present work suggests that sporangia of P. cinnarnorni may have this ability. In all soils, some sporangia germinated directly, others indirectly and some did not germinate during the experiments. This suggests that the microenvironment or the intrinsic properties of individual spot-angia are important in determining behaviour. These experiments demonstrated that sporangia and chlamydospores can be produced in a range of soils both in the presence and absence of an added food source. Since both sporangia and chlamydospores developed from washed mycelium in water without a root disc. it is apparent that the vegetative mycelium contained sufficient metabolites for their formation (without a requirement for exogenous nutrients). Nutrient depletion has been shown to be an important factor in sporangium production (Chen and Zentmyer, 1969) and may have caused a shift from the vegetative to the reproductive phase. The slower and less abundant production of sporangia in soil 4 when compared to the other four soils occurred also in a leachate of this soil. This may have been due to fewer bacteria in this soil able to stimulate sporangium production. A similar suggestion has been made to explain why “suppressive” soils in Queensland yield less sporangia of P. cinnamomi than other “nonsuppressive” soils (Broadbent and Baker, 1973). However, the fact that abundant sporangia were produced in the water control with a root disc suggests that soil 4 may have been actively inhibitory to sporangium development rather than just lacking stimulatory bacteria. Experiment (b) indicated that chlamydospores can be produced over a range of soil moisture regimes. Sporangia and zoospores were. however, observed

cirmamomi

23

only at the highest moisture level (pF ca. 2.6) and in the water control. This indicated that sporangia are produced only in soils of relatively low pF, in this case (soil 1) a little below field capacity (pF ca. 2.4). It could be argued that either a high absolute moisture content or a water content close to field capacity was the significant factor in stimulating sporangium production in this soil. That this is not so is shown by the fact that in the other soils (experiment a) sporangia occurred with moisture contents ranging from 5.9 to 50 per cent and percentages of field capacity from 23.2 to X9 (Tables 2 and 3). However, the pF values of these soils had been adjusted to nearly the same value (2.6). It thus appears that soil water suction pressure rather than absolute water content is the parameter controlling sporangium production by P. cinnamomi in soil at 22°C. This has very important implications in relation to diseases caused by P. cinnamomi in different soils under natural conditions since zoospores are probably the primary agents of root infection. For example. a relatively light shower of rain could produce a low enough suction pressure to allow sporangium production followed by zoospore release in sandy soil. whereas a similar or heavier shower might not be sufficient to induce their formation in a clay soil. So with a similar distribution and intensity of rainfall, infection would occur on fewer occasions in the clay than in the sandy soil, although vegetative growth of P. cirw~arnorni might becomparable in both soils. Other factors, such as drainage from the soil, must be important in influencing the pF and therefore sporangium production. Acknowledgemr,its-This work forms part of a thesis presented for the degree of Ph.D. in the University of Surrey carried out during the tenure of a NATO studentship at the Swiss Federal Research Station, WHdenswil and a NERC studentship at the University of Surrey. I would like to thank Dr. R. M. Jackson and Dr. H. Schiiepp for their advice and helpful discussion throughout the work.

REFERENCES

BLACKWELL E. M. (1943) The life history of Phytophthora cactorum.Trans. Br. mycol. Sot. 16, llLX9. BROADBENTP. and BAKER K. F. (1973) Soils suppressive to Phytophthora root rot in Eastern Australia. (Abstr.) 211~1 International Congress of Plant Pathology. Abstr. no. 0838. CHEN D. W. and ZENTMYERG. A. (1969) Production of sporangia by Phytophthora cinnamomi in axenic culture. Mqcologia 62, 397-402. DENNIS C. and WEBS~R J. (1971) Antagonistic properties of species-groups of Trichoderma-III: Hyphal interaction. Trans. Br. mycol. Sot. 57, 363-369. HENURIX F. F. JR. and KUHLMAN E. G. (1965) Existence of Phytophthora cinnamomi as chlamydospores in soil. (Abst.) Phytopathology 55, 499. KOUYEAS V. (1964) An approach to the study of moisture relations of soil fungi. PI. Soil 20, 35 l-363. KLJHLMAN E. G. (1964) Survival and pathogenicity of Phytophthora cinnamomi in several western Oregon soils. Forrsf Sci. 10, 151&15X.

24

~OSI.MAK>

J. Rt I.VI.S