Control by arbuscular endomycorrhizae of Pratylenchus brachyurus in pineapple microplants

Pratylenchus brachyurus (Godfrey) Filip & Schurr-Steekh. has been reported in association with pineapple roots and is considered as an important pathogen on pineapple. Microplants of Queen Tahiti, Smooth Cayenne and Spanish varieties were inoculated with Glomus sp. (LPA2I) and/or P. brachyurus at transplanting from axenic conditions or one month later. The presence of the nematode did not affect shoot growth of endomycorrhizal plants. Late P. brachyurus inoculation did not influence growth of nonmycorrhizal plants while early pathogen application caused reductions in nonmycorrhizal plant growth. Nematode number per g of root was significantly decreased for endomycorrhizal plants when pathogen was intro- duced at outplanting or one month later. Nematode inoculation affected endomycorrhizal colonization estimated by non vital staining for the Queen Tahiti and Spanish varieties but did not alter development of metabolically active arbuscules in roots of the three varieties. P concentration of endomycorrhizal shoots was higher for all treatments and P. brachyurus tended to decrease mineral concentra- tion of nonmycorrhizal


Introduction
Arbuscular endomycorrhizal fungi (AMF) and soilborne pathogens occur together in the rhizosphere around plant roots. Arbuscular endomycorrhizae can positively influence plant development by improving mineral nutrition, water uptake, hormone production, resistance to root pathogen or tolerance to pesticides (Gianinazzi et al. 1982). Guillemin et al. (1991) have shown the benefits to the growth of pineapple microplants of inoculation with endomycorrhizal fungi.
Nematodes are considered an important factor in reducing pineapple production. Pratylenchus brachyurus (Godfrey) Filip & Schurr-Steekh. causes widespread damage in pineapple plantations, particularly in the Ivory Coast. It was described for the first time in pineapple roots by Godfrey (1929) in Hawaii. This nematode penetrates the elongation zone of roots and develops in the cortical tissue and vascular cylinder (Guérout 1975). Secondary roots can then be destroyed by nematode infestation giving a root system that is essentially composed of primary roots (Caswell et al. 1990). This endopathogen also modifies vegetative plant growth, with reductions in leaf area, and causes a delay in shoot development (Lacoeuilhe andGuérout 1976, Keetch 1982). Decreases in fruit yield can be in the region of 30 to 35% and the number of suckers may be reduced to 80% (Lacoeuilhe andGuérout 1976,Keetch 1982). P brachyurus has a high impact in the Ivory Coast (Guérout 1975), because pineapple is often grown in soil with a pH adapted to nematode proliferation (pH 5 to 5.5) (Sarah 1991).
The potential of AMF to alleviate nematodeinduced plant stress has been previously investigated in different plant species but variable hostplant responses to pathogen-endomycorrhiza interactions have been reported (e.g. Bagyaraj et al. 1979, Kellam and Schenck 1980, Cason et al. 1983, Elliot et al. 1984, Cooper and Grandison 1986, Smith 1987. Ingham 1988, Thomas et al. 1989). In the present work, we have tested interactive effects of AMF and P.brachyurus on plant growth, endomycorrhizal infection development and root-colonising nematode populations of micropropagated pineapple.

Material and methods
Three micropropagated pineapple varieties (Ananas comosus (L.) Merr., Queen Tahiti, Smooth Cayenne (clone CYO) and Spanish varieties) were tested. Plants were raised in a growth chamber under simulated tropical conditions (300pE s ' nr 2 , 29-25°C, I2h day and 70%-90% relative humidity) in a y-irradiated (lOkGy) acid soil (pH 5.0). Pineapple microplants were inoculated with root fragments of Tephrosia ehlenbergiana infect-ed with an isolate of Glomus sp. (LPA2I) in trays containing a soihgravel (1:1, v:v) mix during a four-week acclimatization period (M) (Guillemin et al. 1991) or when one month-old weaned microplants were transplanted individually to pots (l+M). Each pot contained 400 g of the soihgravel mix and was watered daily with distilled water and weekly with 2x20 ml of Hoagland n°2 solution (Hoagland and Arnon 1950) without phosphate.
Inoculation of Pratylenchus brachyurus was performed with about 100 nematodes per microplant at outplanting from axenic conditions (Nematode) or one month later at the end of the weaning period (Nematode+l).
After 3 months in pots, several growth parameters were evaluated: leaf area (cm 2 ), shoot and root fresh mass (g) and shoot dry mass (g) and the N, P, K, Ca and Mg concentrations of shoots determined (Warner and Jones 1967, Comité Inter Instituts pour le diagnostic foliaire 1968,1972). Endomycorrhiza development was estimated microscopically by the method of Trouvelot et al. (1986) after clearing roots and staining fungal tissue with trypan blue (Philipps and Hayman 1970), or for succinate dehydrogenase (SDH) (Smith and Gianinazzi-Pearson 1990) or alkaline phosphatase (ALP) (Tisserant et al. 1993) activities. Arbuscule frequency (A%) was also estimated and the proportion of living and functional arbuscules calculated as mentioned below: P. brachyurus in roots was extracted using the non-destructive procedure described by Sarah (1991).
All treatments were tested in the same experiment for Smooth Cayenne and Spanish varieties however two experiments were done for Queen Tahiti variety. Each treatment consisted of 5 rep-A % after staining of SDH activity Proportion of living arbuscules = A % after staining with trypan blue A % after staining ofALP activity Proportion of functional arbuscules = A % after staining with trypan blue licates and statistical analysis of data was performed using Newman-Keuls test following ANOVA.

Results
Both early (M) and late (I+M) inoculation with the AMF significantly increased the growth of plants compared with nonmycorrhizal controls whether they were infested or not with P. brachyurus (Tables 1 and 4).

Endomycorrhiza inoculation at transplanting from axenic conditions
With the single exception of Queen Tahiti variety root growth, both shoot and root growth of nonmycorrhizal microplants were significantly reduced when nematodes were introduced at the beginning of the acclimatization period. However, shoot growth of the three pineapple varieties was not significantly modified by nematode infestation in plants inoculated with the Glomus sp. at outplanting from axenic conditions (Table 1). Growth reductions due to the nematode were very limited and only occurred for leaf area (Spanish variety) and roots (Smooth Cayenne and Spanish varieties).
Nematode number per g. of root was significantly lower in endomycorrhizal than nonmycorrhizal roots for all three pineapple varieties (Table 1). Timing of P. brachyurus inoculation did not affect nematode numbers developing in the Queen Tahiti and Smooth Cayenne varieties, but later inoculation did reduce pathogen infestation in the Spanish variety (Table 1).
Nematode infestation caused a reduction in P uptake by nonmycorrhizal plants, particularly for the Smooth Cayenne and Spanish varieties, but not in endomycorrhizal plants (Table 2). For the Spanish variety, N, Ca and Mg concentrations of nonmycorrhizal plants were also decreased by nematode infestation at transplanting. However, in endomycorrhizal plants P, Ca and Mg concentrations in shoots of the three varieties inoculated with the nematodes were comparable to those of endomycorrhizal plants growing in the absence of nematodes (Table 2).
Nematode application at outplanting significantly reduced arbuscule frequency (A%) for the Queen Tahiti and Spanish varieties after trypan blue staining (Table 3) but this reduction disappeared with pathogen inoculation one month later. This showed that it was of greatest benefit to establish endomycorrhizal colonization as soon as possible. Reduction of A% was also observed after staining for SDH and ALP activities in the Queen Tahiti variety ( Table 3). The proportion of living and functional arbuscules was not altered by nematode infestation (Table 3).

Endomycorrhizal inoculation at outplanting to pots
Growth of endomycorrhizal plants was significantly greater than of nonmycorrhizal plants (Table 4). Inoculation with P. brachyurus at outplanting reduced the growth of nonmycorrhizal but not endomycorrhizal plants (Table 4).
The nematode population was reduced in endomycorrhizal plants with both timings of the pathogen application for the Queen Tahiti variety (Table 4) but this reduction was observed for the Smooth Cayenne and Spanish varieties only when nematodes and endomycorrhizae were inoculated simultaneously. Endomycorrhiza development after nematode application did not influence nematode infestation of roots of the Smooth Cayenne and Spanish varieties. Timing of pathogen application influenced its presence in the roots of the Spanish variety; indeed the population was greater when the pathogen was introduced before inoculation with the AMF.
Nematodes negatively affected P uptake by nonmycorrhizal but not endomycorrhizal plants (Table 5). Endomycorrhiza formation enhanced Ca and Mg contents of the Queen Tahiti and Spanish varieties and N concentration for the Spanish variety. K contents were higher for the nonmycorrhizal plants.
P. brachyurus application at outplanting sig-  tion at transplanting to pots can compensate growth reductions of plants inoculated with nematodes at outplanting from axenic conditions. Micropropagated plantlets could tolerate better the nematode inoculation at outplanting from axenic conditions followed one month later by endomycorrhizal inoculation than both symbiotic and pathogen inoculations at the beginning of the acclimatization period. The application of nematodes at outplanting from axenic conditions without endomycorrhizal inoculation significantly reduced plant growth but this effect was not observed when plants were infested by nematodes one month later. This supports previous observations that older plants can tolerate pathogen infestation better than younger plants (Cooper and Grandison 1986).
Pathogen effects on endomycorrhizal colonization estimated after non vital staining varied with the pineapple variety. When nematodes were applied at transplanting this significantly reduced arbuscule frequency (A%) in the Queen Tahiti and Spanish varieties. The ability of nematodes to reduce endomycorrhizal development has also been observed by several authors (e.g. O ' B annon andNemec 1979, Elliot et al. 1984), and it has been suggested that nematodes could induce an unfavourable environment for infection by the fungal symbiont (Thomas et al. 1989). Although nematode infestation negatively influenced val- Table 3: Arbuscular frequency observed after staining with trypan blue (TB), succinate dehydrogenase (SDH) or alkaline phosphatase activities and proportion of living (SDH/TB) and functional (ALP/TB) arbuscules of endomycorrhizal pineapple roots at outplanting from axenic conditions (M) and at transplanting to pots (l+M): nematode uninoculated (Control), inoculated (Nematode) and inoculated one month later (Nematode+l). ues for A% of Queen Tahiti variety estimated by SDH and ALP activities, P. bmchyurus did not affect the proportion of living and functional arbuscules of the three pineapple varieties, and consequently did not influence the efficiency of the symbiosis for pineapple. This could partly explain the lack of effect of P. bmchyurus on the growth of endomycorrhizal plants.

Frequency of arbuscules (A%) detected by staining for
Several reports have shown that endomycorrhizal colonization decreases nematode populations in root systems (e.g. Bagyaraj et al. 1979, Saleh andSikora 1984), and Smith et al. (1986) showed that AMP can enhance plant tolerance to nematodes in field conditions. In this study, numbers of nematodes were also significantly reduced in the roots of endomycorrhizal pineapple of the three varieties in comparison to nonmycorrhizal plants, whether nematodes were applied simultaneously with or after the AMP. Reductions in nematode infection have been attributed to modifications in plant physiology caused by the symbiotic fungi. AMP are able to ensure an adequate P nutrition in presence of nematodes and since P is considered as an important factor in plant tolerance (Smith and Kaplan 1988), higher concentrations of this element in endomycorrhizal tissues could have a direct action reducing nematode numbers in roots (MacGuidwin et al. 1985). Changes in root exudates may also alter root attractiveness for nematodes (MacGuidwin et al. 1985), or induce physical and chemical barriers to root penetration (Kellam and Schenck 1980). Table 4: Leaf area (LA), shoot (SFM) and root (RFM) fresh mass, shoot dry mass (SDM) and number of nematodes per g. of roots (Nem root) of nonmycorrhizal (NM) and endomycorrhizal pineapple at transplanting to pot (l+M): nematode uninoculated (Control), inoculated (Nematode) and inoculated one month later (Nematode+l). Endomycorrhizal colonization could also represent a competition for photosynthates in roots (Smith 1987), and thus produce a less favourable environment for the nematodes (Kellam and Schenck 1980) or influence the quality of food reserves of nematodes (MacGuidwin et al. 1985). Endomycorrhizal plants have higher sugar contents, modified hormone balance and modifications in the composition of amino acids (e.g increases in serine and phenylalanine which are nematicidal) (Suresh et al. 1985). Presence of a fungal symbiont in roots can affect the normal life cycle of nematodes (Cason et al. 1983) and reduce nematode size (Sitaramaiah and Sikora 1982).
Other micro-organisms such as bacteria and fungi are also considered as antagonists to nematodes (Cayrol et al. 1992), and the combination of AMF with one or several antagonists could produce a more beneficial synergistic action on plant protection and growth. Fallow could be also used to combat nematode populations in soil (Stirling and Nikulin 1993), but this approach risks decreasing endomycorrhizal potential and reducing soil fertility (Sarah 1987) unless inoculation with efficient AMF after fallow is ensured. The control of nematodes in pineapple, which avoids excess use of nematicides, clearly requires an integrated approach. The results reported here suggest that endomycorrhizae, which are not affect-