Inoculation of containerized (Miller) seedlings with basidiospores of Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) Th M Fr and collinitus (Fr) O Kuntze P Torres, M Honrubia

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P Torres, M Honrubia. Inoculation of containerized Pinus halepensis (Miller) seedlings with basid- iospores of Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) Th M Fr and Suillus collinitus (Fr) O Kuntze. Annales des sciences forestières, INRA/EDP Sciences, 1994, 51 (5), pp.521- 528. ￿hal-00882967￿

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Inoculation of containerized Pinus halepensis (Miller) seedlings with basidiospores of Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) Th M Fr and Suillus collinitus (Fr) O Kuntze

P Torres, M Honrubia

Departamento de Biología Vegetal (Botánica), Facultad de Biología, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain

(Received 5 April 1993; accepted 31 March 1994)

Summary — Pinus halepensis Miller seedlings grown in containers were inoculated with 3 different basidiospore concentrations of Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) Th M Fr and Suillus collinitus (Fr) O Kuntze, in sterile and unsterilized substrate. Six months after germi- nation, the seedlings were evaluated for ectomycorrhizal development and fungal species were isolated from any synthesized. Height, dry weight and percentages of ectomycorrhizas were recorded. There were no significant differences between the 3 inoculated fungal species used on the seedling growth. The highest mean values of height, dry weight and percentage of ectomycorrhizas were obtained with seedlings inoculated with Pisolithus arhizus in sterile substrate. inoculation / basidiospore / I ectomycorrhizas / Pinus halepensis / Pisolithus arhizus / Rhizopogon roseolus / Suillus collinitus

Résumé— Inoculation de plantules de Pinus halepensis Miller, cultivées en contenants, dans des substrats stériles et non stériles, avec 3 concentrations sporales de Pisolithus arhizus (Pers) Rauschert, Rhizopogon roseolus (Corda) Th M Fr et Suillus collinitus (Fr) O Kuntze. Six mois après la germination, les espèces fongiques ont été isolées à partir des ectomycorhizes syn- thétisées. La hauteur, le poids secs et les pourcentages d’ectomycorhizes ont été déterminés. On n’a pas mis en évidence de différences significatives entre les 3 espèces fongiques étudiées sur la crois- sance des plantules. Cependant, les meilleurs résultats en termes de croissance et d’infection myco- rhizienne ont été obtenus sur substrat stérile avec Pisolithus arhizus. inoculation / basidiospore / ectomycorhizes / Pinus halepensis / Pisolithus arhizus / Rhizopo- gon roseolus 1 Suillus collinitus INTRODUCTION adapted to the semiarid conditions of this zone. The controlled mycorrhizal infection of P arhizus, which forms large fruit bodies seedlings intended for use in afforestation containing many basidiospores, is an excel- schemes is not a common practice in Span- lent example of an ectomycorrhizal species ish nurseries. Although the use of soils from adapted to adverse conditions and has a wide of host The established plantations does ensure a range plants (Marx, 1977). degree of infection by ectomycorrhizal fungi, other 2 species used are very common in the seedlings tend to be colonized by fungi P halepensis forests, and their fruit bodies are found in under these adapted to nursery conditions and these large quantities trees. These form probably disappear once they are planted species ectomycorrhizas in vitro with P et out. Furthermore, the use of natural soil as halepensis (Torres al, 1991; Torres and Honrubia, and have a an inoculum exposes the nursery to possi- 1994b) of viable and active basidio- ble infection by pest or pathogens (Molina, high percentage 1977). in slurries obtained from fruit bodies (Torres and Honrubia, 1994a). The use of ectomycorrhizal The present study had 3 objectives. First, spores is the simplest and most economic to examine whether the inoculation of soil method for the inoculation of large numbers with basidiospores of P arhizus, R roseo- of since can be incorporated seedlings they lus and S collinitus is effective for the devel- in the water used for irrigation. However, it opment of ectomycorrhizas. Secondly, to is first necessary to ascertain whether the isolate the fungal species from the ectomy- spores are viable and capable of germinat- corrhizas obtained after inoculation to see ing in the rizosphere of the seedlings to be whether the synthesis established corre- inoculated (Miller et al, 1993; Torres and sponded to the fungus. Thirdly, to deter- Honrubia, 1994a). mine the effect of different concen- During the last 20 years, many success- trations on seedling development in sterile ful experiments have been carried out to and unsterilized substrates. inoculate seedlings with basidiospores of specific fungal species and many authors have used these propagules for the forma- MATERIALS AND METHODS tion of ectomycorrhizas in different species of (Marx and Ross, 1970; Theodorou, Sherwood-type Trioum root-trainers containers 1971, 1984; Theodorou and Bowen, 1973; (175 ml capacity) were used after being steril- Marx, 1976; Hodson, 1979; Marx et al, 1979; ized in water and bleach (1:1). The containers Ruehle, 1980; Alvarez and Trappe, 1983; were filled with 2 types of substrate: 1 ) sterile Beckjord et al, 1984; Marx and Bell, 1985; peat, soil and vermiculite (1:1:1 v/v/v); or 2) unster- ilized soil and vermiculite (1:1:1 v/v/v). The Marx et al, peat, 1989). soil used in both cases came from a local refor- In the present study, 3 fungal species ested pine forest of approximately 20 years stand- were chosen for the basidiospore inoculation ing. Substrate 1 was steam-sterilized 3 times at of Pinus halepensis Miller (Aleppo pine) 100°C for 1 h (once a week for 3 weeks). The substrate in both cases was Pisolithus arhizus pH approximately seedlings: (Pers) 6.5 (in water). Rauschert, Rhizopogon roseolus (Corda) The P halepensis seeds came from the El Th M Fr and Suillus collinitus O Kuntze, (Fr) Valle nursery belonging to the Servicio de Montes in all of which grow naturally Aleppo pine de la Agencia Regional para el Medio Ambiente forests of SE Spain and are therefore well y la Naturaleza de la Región de Murcia. They underwent no prior scarification or stratification tie was clearly observed. If mantle was not clear treatment before germination. or not present mycorrhizal colonization was deter- mined cross-sections and The seeds were rinsed in tapwater and then making examining surface sterilized with 30% H2O for 20 min. After microscopically for the presence of a Hartig net. without mantle and/or net sterilization they were sown in the containers Tips appearing Hartig (approximately 5 seeds/cavity). Germination took were not counted. For the dry weight measure- place at 10-15 d and then all cavities were ments, the seedlings were dried at 65°C for 16 h. thinned to 1 seedling. All data were subjected to analysis of variance Fruit bodies of P arhizus came from P and significant differences was carried out halepensis plantations in El Valle (Murcia). The between the means using a Duncan’s test (P ≤ basidiospores were suspended in sterile distilled 0.05) (Duncan, 1955). water with Tween 80.

The inocula of R roseolus and S collinitus were prepared according to the method described by Isolation of fungal symbionts Castellano and Molina (1989), from fruit bodies from ectomycorrhizas collected in Aleppo pine plantations in El Valle (Murcia). This method consists of preparing spore slurries from fragments of , which are In order to check which fungi are present in the then triturated in sterile distilled water. Spore con- root systems, ectomycorrhizas were isolated. centration in the final solution was calculated with These had previously been charac- a hemacytometer. terized morphologically (ramification, colour, man- The slurries were stored for 10-15 d at 3-4°C tle surface, mycelial strands, etc) using our pre- vious of in vitro before use. Spore viability and activity has been knowledge synthesized examples shown to decrease considerably after 30 d of cold as a basis (Torres et al, 1991; Torres and Hon- storage (Torres and Honrubia, 1994a). rubia, 1994b). Mycorrhizal roots were taken from 15 randomly selected plants. These were surface sterilized and placed in petri dishes with MMN medium (Marx, Experimental design 1969). Sterilization was carried out as follows. The mycorrhizal roots were vigorously washed in a Three spore suspensions of different concentra- solution of 0.01 % Tween 80 to eliminate soil par- tions were prepared for each fungal species: 106, ticles. They were washed in sterile distilled water 4 x 106 and 107 basidiospores/ml. For each ecto- for 30 min and then surface sterilized with 30% mycorrhizal fungus one container with sterile and H2O for 30-40 s. Finally, they were once again another with unsterilized substrate for each spore washed in sterile distilled water. concentration were prepared (6 treatments). After isolation, the mycelia were compared Four inoculation batches were made at 15 d with those of the fungal species used as inoculum intervals following seed germination. The final obtained from fruit body tissues. In addition to quantity of basidiospores per seedling was 4 x macroscopic characterization of the mycelia, their 1.6 x 106 and 4 x 107 in each treatment. 106, microscopic characteristics were examined (clamp Control seedlings in each substrate received connections, ramification, size, pigmentation, etc). no inoculation. The experiment started in Decem- ber and finished in May, using greenhouse con- ditions with a natural and temperature light cycle. RESULTS The plants were watered once or twice a day as necessary. Six months after germination, 15 seedlings Tables I-VI show the mean values for the were randomly selected from each of the 6 treat- height, dry weight and percentage of ecto- ments and from the control The groups. height mycorrhizas of the 15 randomly selected and dry weight of top and root were recorded. from each These are fol- The percentage of ectomycorrhizas was calcu- seedlings group. lated by counting the infected and uninfected tips. lowed by a letter according to the result of Tips were considered as mycorrhizal when man- Duncan’s test.

From tables I and II, which correspond concentration is not reflected in the actual to the seedlings inoculated with P arhizus, it proportion of mycorrhizas developed. can be seen that there are no significant dif- The values obtained for control seedlings ferences between height and dry weight of in sterile substrate differ from those of the 3 inoculated with the 3 con- seedlings spore spore inoculum concentrations used and no centrations in sterile the substrate, although ectomycorrhizas were found in the root sys- difference is highly significant between these tems. seedlings and the uninoculated control ones. In unsterilized soil, there were no signif- The percentage of P arhizus mycorrhizal icant differences in height between the con- colonization is with lower con- higher spore centrations of basidiospores used. As centrations. It is therefore that a possible regards dry weight, however, the first treat- threshold level of spore concentration exists, ment (lowest spore concentration) differs which increase in the number of beyond any from the other 2, these values being higher has a negative effect on myc- propagules than with greater spore concentrations. The orrhizal and total root percentage dry weight. percentages of R roseolus ectomycorrhizas As Marx self-inhibition suggested by (1976) are far below those obtained in sterile soil, may exist at high spore concentrations. the highest values occurring with the highest In the unsterilized substrate, however, concentration of basidiospores. There are there are clear differences between the also a high number of ectomycorrhizas smallest spore concentration and the other developed by native symbionts, at a simi- 2 used; the first always provides lower height lar level to that formed by the inoculated and dry weight data. Root ectomycorrhizal fungus. It seems that, unlike the case of P colonization is far below that found in the arhizus, there is no competition between sterilized soil and the appearance of ecto- the introduced fungus and those already mycorrhizas of native symbionts exercises present in the soil used. a competitive effect on the inoculated fun- The control plants in unsterilized sub- gus. strate showed lower height and dry weight As a rule, the control seedlings showed values than those which had been inocu- the lowest height and dry weight values lated, although they form ectomycorrhizas even in the unsterilized soil, where an with the previously described fungal species unidentified species developed ectomycor- which appeared in the unsterilized soil. rhizas, which we will refer to as Suillus-type. The seedlings inoculated with S collinitus In the seedlings in sterile substrate and inoc- (tables V and VI) in sterile substrate show ulated with R roseolus (tables III and IV), significant differences between the data significant differences were observed for obtained with the lowest spore concentration height and dry weight between the small- and the 2 highest; all the values are much est spore concentration and the other 2 con- higher in the first and similar in the other 2. centrations used. These data are highest The appearance of a possible negative in the lowest concentration and practically effect at high spore concentrations, as equal to the 2 higher concentrations. As in occurred with the other 2 species, is also the previous case, there could well be a observed, although the S collinitus ectomy- threshold level above which an increase in corrhizas percentages do not support this spore numbers might have a negative effect hypothesis. The highest percentage of ecto- on plant growth. The percentages of R rose- mycorrhizas is found with the intermediate olus ectomycorrhizas showed no difference spore concentration; the other 2 concen- between the 3 concentrations. This hypo- trations are similar with no significant dif- thetical negative effect produced by height ferences between them. The control seedlings showed no ecto- The corresponding mycelia were obtained mycorrhizal infection and mean heights and from the mycorrhizas synthesized in vivo, dry weights were below those of the inocu- which were identical to those synthesized lated plants. in vitro for each of the inoculated fungi. The were isolated in MMN Significant differences were observed in ectomycorrhizas the unsterilized soil between the height and medium and the mycelia which developed top dry weight of seedlings inoculated with were compared with those in our collections obtained from the fruit bodies of the corre- the highest spore concentration as opposed The char- to the other 2 treatments; the values with sponding species. macroscopic acteristics of the colonies and the micro- the higher spore concentration had signifi- characteristics of the were cantly higher dry weights. However, there scopic hyphae identical in all cases. This confirms innu- was no difference in the 3 treatments as merable previous studies showing that the regards the mean dry weight of the roots. use of as inoculum is effec- The percentages of ectomycorrhizas formed basidiospores tive for carrying out controlled inoculation, by S collinitus were below those obtained especially if the substrate is sterilized before- in sterile substrate. The widescale presence hand. When an unsterilized substrate is of light-brown ectomycorrhizas of an used, the results in all cases suggest that unknown symbiont was noted. These were the degree of ectomycorrhizal colonization is isolated and identified as a possible Suillus higher than in control experiments, species. although the percentage of ectomycorrhizas devel- Although the control seedlings showed oped by native symbionts also rises. ectomycorrhizal infection, they had lower In general terms, there are no dif- and values than the inoc- large height dry weight ferences between the 3 fungal species used. ulated plants. The highest mean values of height, dry weight and percentage of ectomycorrhizas, was obtained in inoculated with P CONCLUSIONS plants arhizus. This agrees with the results of Ruehle et al (1981), who found that P All the seedlings inoculated with P arhizus, halepensis seedlings inoculated with P R roseolus and S collinitus developed ecto- arhizus mycelia showed higher values for mycorrhizas of identical characteristics to height, fresh weight and root collar diameter those described by Torres et al (1991 ) and than those inoculated with Thelephora ter- Torres and Honrubia (1994b). The uninoc- restris or an uninoculated control. The lat- ulated seedlings grown in sterile substrates ter fungus is very infective in sterile substrate showed no ectomycorrhizal infection, while but can be displaced by native species when those grown in unsterilized substrates unsterilized substrate is used. Ruehle (1980) formed ectomycorrhizas with unknown sym- mentioned somewhat similar results obtained bionts. These mycorrhizas were morpho- with P tadea inoculated with P arhizus. The logically similar to those formed by the lowest levels of ectomycorrhizas developed genus Suillus, which was to be expected by P arhizus in this study occurred when since the soil used came from forests where they were colonized by T terrestris, which is species of this genus predominated. These a very common species in nursery beds. Suillus-type ectomycorrhizas were also The use of sterilized substrate delays observed in the inoculated seedlings grown recolonization by competitors and antago- in unsterilized substrates along with those nists of the fungal species used. The most formed by the inoculated fungus. noteworthy results are those which refer to the dry weight of the roots, which was much Marx DH (1977) Tree host range and world distribution higher in sterilized than in unsterilized sub- of ectomycorrhizal fungus Pisolithus tinctorius. Can J Bot 23, 217-223 strate. This points to an increase in the Marx DH, Bell W (1985) Formation of Pisolithus ecto- of the root a development system, very mycorrhizae on loblolly pine seedlings with spore important characteristic when dealing with pellet inoculum applied at different times. USDA For seedlings which are to be used in revege- Res SE-249, 7p tation programmes in arid and semiarid Marx DH, Ross EW (1970) Aseptic synthesis of ecto- mycorrhizae on Pinus tadea with basidiospores of areas. In conclusion, the of care- importance Thelophora terrestris. Can J Bot 48, 197-198 controlled inoculation in the fully greenhouse Marx DH, Mexal JG, Morris WG (1979) Inoculation of and forest nurseries and the use of sterile nursery seedbeds with Pisolithus tinctorius spores substrates for growth containers, in these mixed with hydromulch increases ectomycorrhizae programmes is confirmed by the results and growth of loblolly . South J Appl For 3, 175- 178 obtained in this experiment. Marx DH, Cordell CE, Maul SB, Ruehle JL (1989) Ecto- mycorrhizal development on pine by Pisolithus tinc- torius in bare-root and container seedling nurseries. ACKNOWLEDGMENTS II. Efficacy of various vegetative and spore inocula. New For 3, 57-66 Miller SL, Torres P, McLean TM (1993) Basidiospore This research was supported by Icona-Lucdeme viability, dormancy, activation and germination in (Ministerio de Agricultura) and a grant from the ectomycorrhizal and saprophytic basidiomycetes. Spanish Ministerio de Educación y Ciencia. Mycol Res97, 141-149 Molina R (1977) Ectomycorrhizal fungi and forestry prac- tice. In: Mushrooms and Man. An Interdisciplinary REFERENCES Approach to (Walters T, ed), Forest Ser- vice, USDA, 147-161 Ruehle JL (1980) Inoculation of containerized loblolly Alvarez IF, Trappe JM (1983) Effects application rate pine seedlings with basidiospores of Pisolithus tinc- and cold soaking pretreatment of Pisolithus tinctorius torius. 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