Báo cáo khoa học: "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"

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Nội dung Text: Báo cáo khoa học: "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"

Original article 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 ectomycorrhizas 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. ectomycorrhizas/ Pinus halepensis / Pisolithus arhizus / Rhizopogon inoculation/ basidiospore / I 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. halepensis / Pisolithus arhizus / Rhizopo- inoculation / basidiospore / ectomycorhizes / Pinus gon roseolus 1 Suillus collinitus
INTRODUCTION to the semiarid conditions of this adapted zone. P arhizus, which forms large fruit bodies The controlled mycorrhizal infection of containing many basidiospores, is an excel- seedlings intended for use in afforestation lent example of an ectomycorrhizal species schemes is not a common practice in Span- adapted to adverse conditions and has a ish nurseries. Although the use of soils from wide range of host plants (Marx, 1977). The established plantations does ensure a other 2 species used are very common in degree of infection by ectomycorrhizal fungi, P halepensis forests, and their fruit bodies the seedlings tend to be colonized by fungi are found in large quantities under these adapted to nursery conditions and these trees. These species form ectomycorrhizas probably disappear they planted once are in vitro with P halepensis (Torres et al, 1991; of natural soil as Furthermore, the out. use Torres and Honrubia, 1994b) and have a an inoculum exposes the nursery to possi- high percentage of viable and active basidio- ble infection by pest or pathogens (Molina, spores in slurries obtained from fruit bodies 1977). (Torres and Honrubia, 1994a). The use of ectomycorrhizal fungus 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 seedlings since they can be incorporated 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 spore 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 pine (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 were filled with 2 types of substrate: 1 ) sterile Ruehle, 1980; Alvarez and Trappe, 1983; peat, soil and vermiculite (1:1:1 v/v/v); or 2) unster- Beckjord et al, 1984; Marx and Bell, 1985; ilized peat, soil and vermiculite (1:1:1 v/v/v). The Marx et al, 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- ing. Substrate 1 was steam-sterilized 3 times at were chosen for the basidiospore inoculation 100°C for 1 h (once a week for 3 weeks). The of Pinus halepensis Miller (Aleppo pine) substrate pH in both cases was approximately seedlings: Pisolithus arhizus (Pers) 6.5 (in water). Rauschert, Rhizopogon roseolus (Corda) The P halepensis seeds came from the El Th M Fr and Suillus collinitus (Fr) O Kuntze, Valle nursery belonging to the Servicio de Montes all of which grow naturally in 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
clearly observed. If mantle was not clear tie underwent prior scarification stratification was no or treatment before present mycorrhizal colonization was deter- germination. not or mined making cross-sections and examining The seeds were rinsed in tapwater and then microscopically for the presence of a Hartig net. surface sterilized with 30% H for 20 min. After O 2 Tips appearing without mantle and/or Hartig net sterilization they were sown in the containers were not counted. For the dry weight measure- (approximately 5 seeds/cavity). Germination took ments, the seedlings were dried at 65°C for 16 h. place at 10-15 d and then all cavities were thinned to 1 seedling. All data were subjected to analysis of variance and significant differences was carried out Fruit bodies of P arhizus from P came between the means using a Duncan’s test (P ≤ halepensis plantations in El Valle (Murcia). The 0.05) (Duncan, 1955). basidiospores were suspended in sterile distilled water with Tween 80. The inocula of R roseolus and S collinitus were to the method described by Isolation of fungal symbionts prepared according Castellano and Molina (1989), from fruit bodies from ectomycorrhizas collected in Aleppo pine plantations in El Valle (Murcia). This method consists of preparing spore In order to check which fungi are present in the slurries from fragments of hymenium, which are root systems, ectomycorrhizas were isolated. then triturated in sterile distilled water. Spore con- centration in the final solution was calculated with These mycorrhizas had previously been charac- terized morphologically (ramification, colour, man- hemacytometer. a tle surface, mycelial strands, etc) using our pre- The slurries were stored for 10-15 d at 3-4°C vious knowledge of synthesized in vitro examples before use. Spore viability and activity has been as a basis (Torres et al, 1991; Torres and Hon- shown to decrease considerably after 30 d of cold rubia, 1994b). Mycorrhizal roots were taken from storage (Torres and Honrubia, 1994a). 15 randomly selected plants. These were surface sterilized and placed in petri dishes with MMN medium (Marx, 1969). Experimental design Sterilization was carried out as follows. The roots were vigorously washed in a mycorrhizal Three spore suspensions of different concentra- solution of 0.01 % Tween 80 to eliminate soil par- tions were prepared for each fungal species: 10, 6 ticles. They were washed in sterile distilled water 4 x 10 and 10 basidiospores/ml. For each ecto- 6 7 for 30 min and then surface sterilized with 30% mycorrhizal fungus one container with sterile and H for 30-40 s. Finally, they were once again O 2 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 106, 1.6 x 106 and 4 x 10 in each treatment. 7 microscopic characteristics were examined (clamp Control seedlings in each substrate received connections, ramification, size, pigmentation, etc). inoculation. The experiment started in Decem- no ber and finished in May, using greenhouse con- ditions with a natural temperature and 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 groups. The height mycorrhizas of the 15 randomly selected and dry weight of top and root were recorded. seedlings from each group. These are fol- The percentage of ectomycorrhizas was calcu- lowed by a letter according to the result of lated by counting the infected and uninfected tips. Duncan’s test. Tips were considered as mycorrhizal when man-
From tablesI 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 seedlings inoculated with the 3 spore con- spore inoculum concentrations used and no centrations in sterile substrate, although the 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 higher with lower spore con- centrations of basidiospores used. As centrations. It is therefore possible that a regards dry weight, however, the first treat- threshold level of spore concentration exists, ment (lowest spore concentration) differs beyond which any increase in the number of from the other 2, these values being higher propagules has a negative effect on myc- than with greater spore concentrations. The orrhizal percentage and total root dry weight. percentages of R roseolus ectomycorrhizas As suggested by Marx (1976) self-inhibition 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 values than those which had been inocu- As a rule, the control seedlings showed lated, although they form ectomycorrhizas the lowest height and dry weight values with the previously described fungal species even in the unsterilized soil, where an which appeared in the unsterilized soil. unidentified species developed ectomycor- 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 from the mycorrhizas synthesized in vivo, mycorrhizal infection and mean heights and which were identical to those synthesized dry weights were below those of the inocu- in vitro for each of the inoculated fungi. The lated plants. ectomycorrhizas were isolated in MMN Significant differences were observed in medium and the mycelia which developed the unsterilized soil between the height and were compared with those in our collections top dry weight of seedlings inoculated with obtained from the fruit bodies of the corre- the highest spore concentration as opposed sponding species. The macroscopic char- to the other 2 treatments; the values with acteristics of the colonies and the micro- the higher spore concentration had signifi- scopic characteristics of the hyphae were cantly higher dry weights. However, there 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 basidiospores as inoculum is effec- The percentages of ectomycorrhizas formed 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, although species. 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 large dif- height and dry weight values than the inoc- ferences between the 3 fungal species used. ulated plants. The highest mean values of height, dry weight and percentage of ectomycorrhizas, was obtained in plants inoculated with P CONCLUSIONS 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 arhizus mycelia showed higher values for R roseolus and S collinitus developed ecto- height, fresh weight and root collar diameter mycorrhizas of identical characteristics to than those inoculated with Thelephora ter- those described by Torres et al (1991 ) and restris or an uninoculated control. The lat- Torres and Honrubia (1994b). The uninoc- ter fungus is very infective in sterile substrate ulated seedlings grown in sterile substrates but can be displaced by native species when showed no ectomycorrhizal infection, while unsterilized substrate is used. Ruehle (1980) those grown in unsterilized substrates mentioned somewhat similar results obtained formed ectomycorrhizas with unknown sym- with P tadea inoculated with P arhizus. The bionts. These mycorrhizas were morpho- lowest levels of ectomycorrhizas developed logically similar to those formed by the by P arhizus in this study occurred when genus Suillus, which was to be expected they were colonized by T terrestris, which is since the soil used came from forests where a very common species in nursery beds. species of this genus predominated. These The use of sterilized substrate delays Suillus-type ectomycorrhizas were also 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
Marx DH (1977) Tree host range and world distribution the dry weight of the roots, which was much of ectomycorrhizal fungus Pisolithus tinctorius. Can higher in sterilized than in unsterilized sub- J Bot 23, 217-223 strate. This points to an increase in the Marx DH, Bell W (1985) Formation of Pisolithus ecto- development of the root system, a very mycorrhizae on loblolly pine seedlings with spore important characteristic when dealing with pellet inoculum applied at different times. USDA For Res SE-249, 7p seedlings which are to be used in revege- Marx DH, Ross EW (1970) Aseptic synthesis of ecto- tation programmes in arid and semiarid mycorrhizae on Pinus tadea with basidiospores of areas. In conclusion, the importance of care- Thelophora terrestris. Can J Bot 48, 197-198 fully controlled inoculation in the 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 and growth of loblolly pines. South J Appl For 3, 175- programmes is confirmed by the results 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 Approach to Mycology (Walters T, ed), Forest Ser- REFERENCES vice, USDA, 147-161 Ruehle JL (1980) Inoculation of containerized loblolly pine seedlings with basidiospores of Pisolithus tinc- Alvarez IF, Trappe JM (1983) Effects application rate torius. USDA For Serv Res Note-291, 4 p and cold soaking pretreatment of Pisolithus tinctorius spores on effectiviness as nursery inoculum on west- Ruehle JL, Marx DH, Abourouh M (1981) Development ern conifers. Can J For Res 13, 533-537 of Pisolithus tinctorius and Thelephora terrestris ecto- mycorrhizae on seedlings of coniferous trees impor- Beckjord PR, McIntosh MS, Hacskaylo E, Melhuish JH tant to Morocco. Ann Rech For Maroc 21, 281-296 Jr (1984) Inoculation of loblolly pine seedlings at plant- ing with basidiospores of ectomycorrhizal fungi in chip Theodorou C (1971) Introduction of mycorrhizal fungi form. Northeast For Exp Sta Res Note NE-324, 4 p into soil by spore inoculation of seed. Aust For 35, 23-26 Castellano MA, Molina R (1989) Mycorrhizae. In: The Container Tree Nursery (TD Landis et al, eds). Agric Theodorou C (1984) Mycorrhizal inoculation or pine Handbook 674, Washington DC, USDA, Forest Ser- nurseries by sprying basidiospores onto soil prior to vice, 102-107 sowing. Aust For 47, 76-78 Duncan D (1955) multiple F-test. and Multiple range Theodorou C, Bowen GD (1973) Inoculation of seeds Biometrics 11, 1-42 and soil with basidiospores of mycorrhizal fungi. Soil Biol Biochem 5, 765-771 Hodson TJ (1979) Basidiospore inoculations of soil: the effect of application timing on Pinus ellioti seedling Torres P, Honrubia M (1994a) Basidiospore viability in development. S Afr For J 108, 10-15 stored slurries. Mycol Res 98 (5), 527-530 Marx DH (1969) The influence of ectotrophic mycor- Torres P, Honrubia M (1994b) Ectomycorrhizal associ- rhizal fungi on the resistence of pine roots to ations proven for Pinus halepensis. Isr J Plant Sci pathogenic infections. I. Antagonism of ectomycor- 42, 51-58 rhizal fungi to root pathogenic fungi and soil bacteria. Torres P, Honrubia M, Morte MA (1991) In vitro syn- Phytopathology 59, 153-163 thesis of ectomycorrhizae between Suillus collini- Marx DH (1976) Synthesis of ectomycorrhizae on loblolly tus (Fr) O Kuntze, Rhizopogon roseolus (Corda) Th pine seedlings with basidiospores of Pisolithus tinc- M Fr with Pinus halepensis Miller. Mycotaxon 41, For Sci 22, torius. 13-20 437-443