Contribution of arbuscular mycorrhizas to biological protection of micropropagated pineapple ( , Ananas comosus ( L . ) Merr ) against Phytophthora cinnamomi Rands

Guillemin, J.P. 1 , Gianinazzi, S.', Gianinazzi-Pearson, V. 1 & Marchal, J.2 1994. Contribution of endomycorrhizas to biological protection of micropropagated pineapple (Ananas comosus (L.) Mere) against Phytophthora cinnamomi Rands. Agricultural Science in Finland 3: 241-251. (‘ Laboratoire de Phytoparasitologie, INRA-CNRS, Station de Génétique et d’Amdlioration des Plantes, INRA, BV 1540, 21034 Dijon Cedex, France and 2 Laboratoire de Physiologie et Biochimie, CIRAD, FLUOR, Avenue du Vai de Montferrand, BP 5035, 34032 Montpellier Cedex 01, France.)


Introduction
In soil, plant roots develop in the presence of micro-organisms, some of which can have a positive (e.g.arbuscular mycorrhizal fungi (AMF)) or negative (e.g.pathogen fungi) impact on plant growth.Root rot of pineapple (Ananas comosus (L.) Merr.), caused by the soilborne fungus Phytophthora cinnamomi Rands, is a major problem in pineapple production (Mehrlich 1936).This pathogen damages root systems, negatively in-fluences shoot development (Mehrlich 1934)  causes the production of fruits without commer- cial interest (Py et al. 1984) and is able to devas- tate plantations (Anderson 1951).The disease is presently controlled by chemical applications (Pegg 1977, Rochbach and Schenck 1985) and/ or by modifying the soil environment before planting (drainage, pH reduction).
Several reports have indicated the bioprotective effect of endomycorrhiza formation against pathogens (Gianinazzi et al. 1982, Paulitz and  Linderman 1991).However results are contra- dictory for P.cinnamomi.Whilst an important de- crease in root rot disease was observed for en- domycorrhizal plants of Chamaecyparis lawsoni- ana L. (Bärtschi et al. 1981), the impact of the disease was not modified by endomycorrhization of avocado (Matare and Hatting 1978) or citrus (Davis et al. 1978).The aim of this work was to evaluate the success of AMF as biological con- trol agents against damage by P.cinnamomi in pineapple plant production.
Plant growth was evaluated by leaf area (Chauvel 1991), shoot and root fresh weight and shoot dry weight.N, P, K, Ca and Mg contents of shoots were analysed (Warner and Jones 1967, Comité   Inter Instituts pour le diagnostic foliaire 1968,  1972).Endomycorrhizal colonization was evalu- ated by the Trouvelot et al. (1986) method (intensity of infection in the root cortex (M%) and arbuscular frequency in the root cortex (A%)) after clearing and staining with trypan blue (Philipps and Hayman 1970) and after staining for succinate dehydrogenase (SDH) (living) (Smith and Gianinazzi-Pearson 1990) or alka- line phosphatase (ALP) (functional) (Tisserant  et al. 1993) activities.
Each treatment consisted of 5 replicates and all data was analysed statistically by Newman- Keuls tests.

Development of the endomycorrhizal infection
The endomycorrhizal colonization was well de- veloped in roots of both pineapple varieties.Eval- uations of infection intensity (M%) were between 83% and 91% after non vital staining with trypan blue (Fig. 1).Values of M% estimated after stain- ing for SDH and ALP activities, to evaluate living and functional infection respectively, were lower, fluctuating between 48% and 63% for the former, and between 27% and 38% for the latter (Fig. IA, 18, 2A, 2B).P.cinnamomi did not significantly affect infection intensity (M%) for either inoculation time, that is at transplanting to pots (Fig. lA, 2A) or one month later (Fig. 18, 2B).
Arbuscule frequency (A%) estimated by trypan blue and ALP staining was significantly reduced for the Queen Tahiti variety in presence of the highest inoculum level of P. cinnamomi at out- planting to pots (Fig. 1C).For the Smooth Cay- enne variety, reduction of A% was observed only for ALP staining and after inoculation of highest concentration of the pathogen at outplanting to pots (Fig. 2C).

Development of P cinnamomi infection
No necroses were observed on roots of non-mycorrhizal and endomycorrhizal plants infected by P. cinnamomi at any concentrations of the pathogen inoculum.

Plant growth
The important development of the AMF in roots of both pineapple varieties was reflected in the better growth of endomycorrhizal plants, with or without P. cinnamomi (Tables 1,2, 3,4).Effect of P. cinnamomi at outplanting to pots P. cinnamomi significantly decreased shoot growth of non-mycorrhizal plants of the Queen Tahiti variety at all dilutions whilst such a negative ef- fect was only observed for endomycorrhizal plants at the highest inoculum level of the pathogen (Table 1).All concentrations of P. cinnamomi significantly decreased root growth of non-mycorrhizal plants whilst root growth of endomycorrhizal plants was only negatively influenced by the two higher levels of pathogen inoculum (Ta- ble 1).Shoot and root growth of endomycorrhizal plants, whether infected or not by P. cinnamo- mi, was always greater than that of non-mycorrhizal plants.
Plants of the Smooth Cayenne variety tolerat- Fig. 1.Intensity of infection (M%) (A and B) and arbuscular frequency (A%) (C and D) observed after trypan blue (TB), succinate dehy- drogenase (SDH) and alkaline phosphatase (ALP) staining of roots of endomycorrhizal Queen Tahiti variety of pineapple inocu- lated with P. cinnamomi at differ- ent dilutions at outplanting to pots (A and C) and one month later (B   and D).Values for each staining followed by different letters are significantly different (p = 0.05).
ed P. cinnamomi better.Only the inoculum dilu- tions of 1:100 and 1:10 affected shoot growth of non-mycorrhizal plants.For endomycorrhizal plants, shoot growth was not significantly affect- ed by the pathogen at any concentration (Table 2).However, root growth of endomycorrhizal plants was altered by higher levels of pathogen inocu- lum, but values were always greater than those of non-mycorrhizal plants, with or without the pathogen.
In these experiments, root/shoot ratios (R/A) of endomycorrhizal plants were always lower than those of non-mycorrhizal plants for both pineap-pie varieties except for endomycorrhizal plants of the Queen Tahiti variety in presence of the high- est level of P. cinnamomi inoculum (Fig. 3A, 3C).
In the absence of P. cinnamomi, endomycorrhiza formation improved shoot mineral contents of the Queen Tahiti variety (Table 5).Pathogen inoculation caused a reduction in the P concen- tration of non-mycorrhizal plants.Decreases in Ca and Mg nutrition were less important for P. cinnamomi-inoculated endomycorrhizal plants.However, N and K concentrations tended to increase in the presence of P. cinnamomi in all plants (Table 5).Fig. 2. Intensity of infection (M%) (A and B) and arbuscular frequency (A%) (C and D) observed after trypan blue (TB), succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) staining of roots of endomycorrhizal Smooth Cayenne variety of pineapple in- oculated with P. cinnamomi at different dilutions at outplanting to pots (A and C) and one month after later (B and D).Values for each staining followed by differ- ent letters are significantly differ- ent (p = 0.05).
Table 1, Leaf area (cm 2 ), shoot (g) and root (g) fresh mass and shoot dry (g) mass of endomycorrhizal (M) and nonmycorrhizal (NM) Queen Tahiti variety of pineapple, inoculated at outplanting to pots with Phytophthom cinnamomi at different dilutions.Values in a column followed by different letters are significantly different (p = 0.05) Table 2. Leaf area (cm 2 ), shoot (g) and root (g) fresh mass and shoot dry (g) mass of endomycorrhizal (M) and nonmycorrhizal (NM) Smooth Cayenne variety ofpineapple, inoculated at outplanting to pots with Phytophthora cinnamomi at different dilutions.Values in a column followed by different letters are significantly different (p = 0.05) Table 3. Leaf area (cm 2 ), shoot (g) and root (g) fresh mass and shoot dry (g) mass of endomycorrhizal (M) and nonmycorrhizal (NM) Queen Tahiti variety of pineapple, inoculated one month after outplant- ing to pots with Phytophthora cinnamomi at different dilutions.4. Leaf area (cm 2 ), shoot (g) and root (g) fresh mass and shoot dry (g) mass of endomycorrhizal Values in a column followed by different letters are significantly different (p = 0.05) Fig. 3. Root/shoot ratios of non- mycorrhizal (NM) and endomycorrhiza!(M) Queen Tahiti (A, B)   and Smooth Cayenne (C, D) vari- eties of pineapple inoculated with Phytophthora cinnamomi at differ- ent dilutions: A, C) at outplanting to pots and B, D) one month later.Values followed by different letters are significantly different (p = 0.05).

Pathogen
Table 5. Mineral concentration (% of dry mass) of shoot of nonmycorrhizal (NM) and endomycorrhizal (M) Queen Tahiti variety of pineapple inoculated with Phytophthora cinnamomi at different dilutions at outplanting to pots For the Smooth Cayenne variety, positive ef- fects of endomycorrhiza on P, Ca and Mg nutri- tion was more important in presence of the pathogen (Table 6).N and K concentrations were not modified by P. cinnamomi inoculation and were generally slightly lower for endomycorrhizal plants (Table  6).

Effect of P. cinnamomi one month after outplanting to pots
In contrast to non-mycorrhizal plants, shoot growth of endomycorrhizal plants of the Queen Tahiti variety was not altered by P.cinnamomi inoculation (Table 3).However, the root growth of both endomycorrhizal or non-mycorrhizal plants of this variety was reduced by the pathogen (Table 3).P.cinnamomi did not affect shoot or root growth of the Smooth Cayenne variety (Table 4).
As could be expected, root/shoot ratios were lower in endomycorrhizal plants, with the exception of the Queen Tahiti variety in presence of the highest level of P. cinnamomi inoculum (Fig. 38, 3D).
Endomycorrhization increased P, Ca and Mg nutrition of the Queen Tahiti variety with and without P cinnamomi (Table 7).However, N con- centration of endomycorrhizal plants was less (phenomenon of dilution).The effect of the symbiotic fungus on P nutrition was more important in presence of the pathogen for the Smooth Cayenne variety (Table 8).For both varieties, N and K contents were lower in endomycorrhizal plants and P. cinnamomi inoculation did not modify this effect (Tables 7,8).

Discussion
Experiments with both pineapple varieties showed that the endomycorrhizal effect on plant growth was not influenced by P cinnamomi, except for the Queen Tahiti variety in the presence of the highest concentration of pathogen, when patho-gen inoculation was carried out at outplanting to pots.The negative effect of P. cinnamomi on non- mycorrhizal plants was likewise important for the Queen Tahiti variety when inoculation was per- formed at outplanting to pots.Pathogen inocula- tion did not alter endomycorrhizal colonization of this variety roots but the highest concentration of inoculum depressed fungal activity (arbuscule formation, ALP activity and endomycorrhizal ef- fect).The reduction in endomycorrhizal plant growth could be explained by a less efficient en- domycorrhizal symbiosis in the presence of P. cinnamomi.
The influence of the pathogen depends on sev- eral factors.Although root necroses were not ob- served both varieties of micropropagated pineapple showed varying susceptibility to negative ef- fects of P. cinnamomi.Better shoot growth of the Smooth Cayenne variety following colonization by AMF was less affected by P. cinnamomi than that of the Queen Tahiti variety.The level of pathogen inoculum influenced plant growth in a similar way to that reported by Davis and Menge  (1981) for citrus, with growth of endomycorrhizal pineapple being decreased at higher levels of P. cinnamomi inoculum.The protective effect of the symbiosis can also change with the AMF.Bärtschi et al. (1981) showed that it was more interesting to inoculate with a mixture of symbiotic fungi to ensure good plant growth and a good level of tolerance towards the pathogen.Such a mixture could contain efficient fungi for both mineral nutrition and protection, and so act synergistically to provide more efficient tolerance to the pathogen.
It is well known that AMF positively influ- ence P nutrition of plants (Harley and Smith  1983) and increases in P nutrition have been sug- gested to decrease root membrane permeability, therefore reducing and modifying root exudation (Ratnayake et al. 1978).Root exudates of endomycorrhizal plants have been reported to con- tain more arginine and reducing sugars (Baltrus- chat and Schönbeck 1975), and changes in exu- date composition can modify rhizosphere populations and decrease pathogen activity (Graham  and Menge 1982).Meyer and Linderman (1986)  reported reductions in sporangia and zoospore production by P. cinnamomi in rhizosphere soil ex- tracts from endomycorrhizal roots.Better absorption of P by endomycorrhizal roots could also counterbalance pathogen damage (Davis and  Menge 1980), but P is probably not the only factor contributing to pathogen tolerance (Graham and Egel 1988).The present study on pineapple shows that the influence of P. cinnamomi also depends on the age of the two varieties at the time of pathogen inoculation and that this is modified by endomycorrhiza infection.Non-mycorrhizal plants were more severely affected by early inoculation with the pathogen but no such difference was observed with endomycorrhiza for- mation, indicating modifications in the physiology of the plant.Endomycorrhiza can influence other aspects of plant physiology than mineral nutrition.As could be expected, they modified biomass distribution in pineapple, root production being lower in relation to that of shoots.However, the application of the highest inoculum level of P. cinnamomi inversed this proportion for Queen Tahiti variety, suggesting that the en- domycorrhizal plants may have produced a more important root system to support the pathogen.AMF can also influence phenol metabolism and root lignification (Dehne and Schönbeck 1979), making plants better adapted to resist to pathogen aggressions.
Other micro-organisms can show a potential for biological control of Phytophthora root rot, such as antagonistic bacteria and fungi (Broad- bent and Baker 1974, Gees and Coffey 1989,  Ownley and Benson 1992).Calvet et al. (1993)  have reported the synergistic action of a fungal antagonist with an AMF in increasing marigold growth in the presence of Pythium ultimum.This effect may be through increases in the population of the antagonistic micro-organism under the influence of AMF (Secilia and Bagyaraj 1987), and opens the possibility of using both groups of mycoflora for improving biocontrol of the root pathogen.

Conclusion
AMF can be considered as potential biological control agents contributing to tolerance to P cin- namomi in pineapple; the Smooth Cayenne vari- ety may be more tolerant to P. cinnamomi ag- gression.It will be interesting to use this bio- technology in association with other antagonists, or with reasonable pesticide applications (Aziz et al. 1990, Guillemin et al. 1993), when pathogen pressure is very important.Futhermore, introduction of endomycorrhization during early stages of pineapple plant production represents an interesting technology towards improving plant development with decreases in chemical input.
Values in a column followed by different letters are significantly different (p = 0.05) Table M) and nonmycorrhizal(NM)Smooth Cayenne variety of pineapple, inoculated one month after outplanting with Phytophlhora cinnamomi at different dilutions. (

Table 6 .
Mineral concentration (% of dry mass) of shoot of nonmycorrhizal(NM)and endomycorrhizal (M) Smooth Cayenne variety of pineapple inoculated with Phytophthora cinnamomi at different dilu-

Table 7 .
Mineral concentration (% of dry mass) of shoot of nonmycorrhizal (NM) and endomycorrhizal (M) Queen Tahiti variety of pineapple inoculated with Phylophthora cinnamomi at different dilutions one month after outplanting to pots

Table 8 .
Mineral concentration (% of dry mass) of shoot of nonmycorrhizal(NM)and endomycorrhizal (M) Smooth Cayenne variety of pineapple inoculated with Phytophthora cinnamomi at different dilu- tions one month after outplanting to pots