PEITSA MIKOLA
Importance and technique of mycorrhizal inoculation
THE MYCORRHIZA, a symbiotic association between fungal hyphae and roots of higher plants, has been recognized since the last century, and mycotrophy has been intensively studied for more than 80 years. The advance of mycorrhizal research, as well as the theories put forward during past decades, have been comprehensively reviewed in a number of textbooks (e.g., Kelley, 1950; Harley, 1959; Lobanow, 1960). A detailed review is, therefore, unnecessary here; it will be enough to recall a few basic features of the phenomenon.
Frank (1885), the great pioneer of mycorrhizal research, already distinguished mycorrhizae of two basically different morphological types, which he called ectotrophic and endotrophic. Although some intermediate types also exist, Frank's classification is still in use today. In a characteristic ectotrophic mycorrhiza the fungus forms a compact sheath or mantle around the rootlet, from which hyphae grow inward to the cortex, forming a continuous network (known as the Hartig net) between the cortical cells, and outward to the surrounding soil. In the endotrophic mycorrhiza, on the contrary, the fungus grows mainly inside the cortical cells, there is no external mantle or intercellur network, and only a few hyphae grow outside the root. In both types the growth of the fungus is restricted to the cortical tissue of the root.
Endotrophic mycorrhizae are widely distributed throughout the plant kingdom and include a great variety of anatomical structures. Thus, for instance, the endotrophic mycorrhizae in the plant families Orchidaceae, Graminaceae, and Ericaceae, and in several broad-leaved trees have different anatomical structures and probably also differ in their physiological functions.
(¹ PEITSA MIKOLA is at the Department of Silviculture, University of Helsinki, Finland. This is a summary report of a research project conducted under an FAO André Mayer fellowship. The author acknowledges the generous financial support by FAO and the excellent cooperation with personnel of forest departments, research institutes and forestry schools, as well as local FAO and United Nations Development Programme representatives, during a study tour through 22 countries in 6 continents.)
Correspondingly, the fungal species involved may belong to different taxonomic groups.
Compared with the endotrophic mycorrhiza, the ectotrophic type has a much more limited distribution and a greater morphological uniformity. It is most characteristic of the families of Pinaceae, Fagaceae and Betulaceae, i.e., the principal tree species of the cool and temperate forests. Later on, ectotrophic mycorrhizae have been found in trees of some other areas too, as, for instance, in the Australian eucalypts (Chilvers and Pryor, 1963, and others) and several tropical species of Cesalpiniaceae (Peyronel and Fassi, 1957; Fassi and Fontana, 1961, 1962) and Dipterocarpaceae (Singh, 1966). The most recent list of plant genera with ectotrophic mycorrhizae has been published by Moser (1967).
The fungal partner of an ectotrophic mycorrhiza is usually a basidiomycete. Some 80 species of Basidiomycetes have been experimentally demonstrated and several times as many are suspected to be mycorrhiza-formers with forest trees (Trappe, 1962).
Whatever the physiological function and ecological importance of the ectotrophic mycorrhizal association, the plain fact is that tree roots in natural forests are almost invariably mycorrhizal, at least in the cool and temperate zones. Although nonmycorrhizal seedlings of pines and other ectotrophs can be grown under experimental conditions, they can hardly be found in nature. Since mycorrhizal association is so universally distributed in the cool and temperate forests, it must be a result of a long evolution and belong to the normal life of the particular trees and ecosystems in question. Thus Singer (1963a), for instance, compares the ectotrophs (trees and their ectotrophic fungal associates) with lichens, in which nonchlorophyllous fungi likewise live in organized symbiosis with green plants.
Mycorrhizae require little attention in the management of natural forests. Mycorrhiza-forming fungi are practically ubiquitous, and mycorrhizae are in all probability formed by the species best suited to the prevailing conditions. There is no need for the introduction of mycorrhizal fungi into the soils and, if new species are introduced, they seem to have few chances to survive in competition with the indigenous fungal population.
Therefore, " 99 percent of all practicing foresters will not have to lose any sleep over the problem of mycorrhizal infection.'' (Wilde, 1944).
In man-made forests the situation is different. Trees are often grown far from their natural range and even on other continents, where the appropriate mycorrhizal fungi may be lacking. If the introduced tree species are mycotrophic, afforestation may fail completely, simply owing to the absence of proper symbionts of the trees. Unsuccessful species trials may result in erroneous conclusions if the trees are not provided with their true symbionts. Thus the story of the introduction of exotic pines to many countries begins with a long succession of failures, until mycorrhizal infection was brought in, in one way or another (Hatch, 1936; Rayner, 1938; Clements, 1941; Briscoe, 1959; Lobanow, 1960; van Suchtelen, 1962; Gibson, 1963; Madu, 1967).
Afforestation activity has grown tremendously in the last 20 years. The FAO Secretariat (1967) has estimated that there are already some 80 million hectares of manmade forests in the world, and in the future some 4 million hectares will be planted every year. The bulk of this planting programme comprises exotic conifers, mainly pines. Development programmes for numerous tropical and subtropical countries include afforestation schemes in which exotic pines play a central role. Mycorrhizal research, however, has not been able to keep pace with the rapidity of practical developments. " The plain fact is that nothing is known for certain as yet." (Parry, 1956). Afforestation has been successful in general, it is true, but certainly many failures could have been avoided if more information on the biology of mycorrhizal fungi and the ecology of their symbiotic association had been available.
In order to promote mycorrhizal research and to coordinate the work done in different countries, a working group was established at the twelfth congress of the International Union of Forest Research Organizations (IUFRO) at Oxford, England, in 1956. At the meetings of this working group the importance of mycorrhizal inoculation in afforestation as well as its practicable techniques were discussed, and it appeared that present knowledge was still very inadequate. As a basis for future research, a survey of the present situation was considered highly desirable and was made possible by FAO, which granted an André Mayer research fellowship to the author.
INTRODUCTION OF EXOTIC TREES WITHOUT MYCORRHIZAL INOCULATION
Numerous examples indicate the necessity of mycorrhizal inoculation at the introduction of new tree species. On the other hand, there are plenty of experiences of the successful introduction and large-scale cultivation of exotic pines and other conifers without any intentional inoculation. In South Africa and Chile, for instance, no trouble due to the absence of appropriate mycorrhizal fungi has ever been experienced:, and even in those countries where mycorrhizal inoculation has later proved useful or necessary, pines had often been grown before anything was known about mycorrhizae. Thus in Western Australia soil for the first inoculation was taken from an old nursery or from under an existing shelterbelt plantation (Kessell, 1927); in other words, pines had been introduced before and grown apparently without inoculation. Likewise in Rhodesia pines were introduced as long ago as 1900 and several thriving plantations were established in subsequent decades (Streets, :1962), although mycorrhizal inoculation was only started and its importance recognized as late as 1928 (Anon., 1931). Similar examples can be taken from many other countries. Even in recent years new nurseries have been established far from older nurseries or existing plantations and good seedlings have been raised without soil inoculation.
Examples of this kind are often taken as evidence that mycorrhizae are not important or necessary to exotic pines. It is most unlikely, however, that any pine can grow in natural surroundings to an age of several years without mycorrhizal association. If sowings or plantations of pine have been successful without inoculation, this does not mean that the trees are non-mycorrhizal. Closer examination has invariably shown the trees to be mycorrhizal. The question then arises, of how the trees have acquired the mycorrhizal infection.
MODES OF TRANSPORTATION ON OF MYCORRHIZAL INFECTION
In most countries, where pines or other ectotrophic trees are not indigenous, mycorrhizal fungi have probably first arrived in the roots of potted plants. According to historians, early settlers often brought trees from their home country and planted them around their new homes (Stephens and Kidd, 1353a; Pryor, 1958). Thus, probably large numbers of living mycorrhizal seedlings of pine, oak, and other European trees have been planted in South Africa, Australia, New Zealand and Latin America as much as two or three hundred years ago. In those days there were no plant quarantine regulations restricting the import of living plants.
Probably mycorrhizal fungi have also arrived in many countries through botanical gardens. According to old records, the first specimens of many exotic trees were brought to the gardens as potted plants.
Mycorrhizal infection may also have arrived as spores attached to imported seed. In tropical and subtropical countries it is customary to extract pine seed in direct sunshine right in the midst of the woods, where the seed can easily become contaminated with both fungal spores and mycorrhizal soil. Acorns are often collected directly from the ground. Formerly there were no quarantine regulations to require disinfection of imported seed. So far no definite proof is available that a mycorrhizal fungus arrived anywhere as spores attached to imported seed. Many observations, however, strongly support such an assumption.
Airborne spore infection must also be taken into consideration. So far very little is known about the viability and germination of the spores of mycorrhizal fungi. It is a well-known fact that spores of many mycorrhizal species are very difficult or even impossible to germinate on synthetic media in laboratory conditions. But, on the other hand, numerous experiences indicate the great ease with which mycorrhizal infection spreads-in fact, to such a degree that special precautions are usually necessary to protect experimentally-grown nonmycorrhizal seedlings from airborne infection. The possibility of spore infection also depends on the longevity of the spores. If spores can remain viable for over a year in dry condition (Chastukhin, 1950), the chances of airborne infection in nurseries are greatly increased. The probability of spore infection decreases, of course, along with increasing distance from existing forests or plantations but, at least theoretically, such a possibility can nowhere in the world be completely eliminated.
It is often impossible to determine with certainty the origin of mycorrhizal infection in nurseries which have not been intentionally inoculated. In countries where inoculated nurseries and plantations already exist, infection may be transported to new ones through spore flight or, for instance, in soil adhering to tools, car tires or shoes of foresters moving from one nursery to another.
INDIGENOUS MYCORRHIZAL FUNGI
Experiences on the necessity of mycorrhizal inoculation have been confined mainly to exotic pines in tropical and subtropical countries and to a lesser extent to other genera of the same family (Larix, Picea, Pseudotsuga) and also to some other mycotrophic trees, such as oaks. Recent studies have shown, however, that many indigenous trees of the respective areas also have ectotrophic mycorrhizae and, accordingly, there are indigenous fungi forming ectotrophic mycorrhizae. Thus, a new question arises as to whether or to what degree these indigenous fungi are able to form mycorrhizal associations with introduced pines or other exotic trees.
Second to Pinaceae, Fagaceae is the best known ectotrophic tree family, all species of which probably have ectotrophic mycorrhizae. Thus, ectotrophic mycorrhizae are known on all the Nothofagus species of New Zealand (Morrison, 1956) and South America (Singer, 1963b, 1964), as well as in the allied family of Dipterocarpaceae (Singh, 1966). The genus Eucalyptus deserves special attention, since pines have been introduced to Australia, the natural range of Eucalyptus, and, on the other hand, eucalypts have been planted on a large scale as exotics in other tropical and subtropical continents. It is also a question of primary interest whether mycorrhizal fungi of the trees of Cesalpiniaceae family can infect pines, since pines are often planted in savanna woodlands, the original forest cover of which mainly consists of species of Cesalpiniaceae.
In general, mycorrhizal fungi are not particularly specialized in regard to their host plants (cf. Trappe, 1962). Many of them form mycorrhizae with several tree species, and even with both conifers and broad-leaved trees. Thus, Amanita muscaria has been experimentally proved to form mycorrhizae with several species of Pinus, Picea, and Betula, and is suspected to do so with other genera too. The most cosmopolitan mycorrhizal species, Cenococcum graniforme, is known to form mycorrhizae with 25 tree genera, including Nothofaqus and Eucalyptus (Trappe, 1964).
The best known ectotrophic mycorrhizal genus of temperate forests is Boletus. Most Boletus species of Europe and North America are symbiotic with forest trees and, consequently, indigenous Boleti of other continents probably are mycorrhizal with some local trees (cf. Palm, 1930) and potential symbionts for introduced exotics too. The same holds true of other mycorrhizal genera, such as Russula, Lactarius, Cortinarius, and Tricholoma (Singer, 1963b).
Anderson (1966) found sporophores of many local fungi (spp. of Amanita, Boletus and Tricholoma) in Eucalyptus plantations in Italy, probably forming mycorrhizae with Eucalyptus. If indigenous Italian fungi can form mycorrhizae with eucalypts, then correspondingly the Australian symbionts of Eucalyptus may be equally able to establish root symbiosis with introduced pines. According to Bowen (1963), Eucalyptus forest soils of South Australia contain indigenous fungi which are able to form mycorrhizae with Pinus radiata, although the population density of these fungi may be low.
Very little is known about the mycorrhizal fungi of the tropical families Cesalpiniaceae and Dipterocarpaceae. The experiments of Olatoye (1966) and others, however, strongly support the theory that indigenous fungi of savannas and rainforests cannot infect introduced pines, at least without some kind of activation.
INTRODUCED MYCORRHIZAL FUNGI
Although mycorrhizal association may possibly be established between introduced trees and indigenous fungi, it is a general experience that mushrooms fruiting in exotic plantations do not belong to the indigenous flora of the respective area.
The history of the arrival of new fungal species in various countries has not been well documented. As was mentioned above, potted seedlings with mycorrhizal roots were often transported from one country to another hundreds of years ago and, consequently, the commonest mycorrhizal fungi were introduced to many countries long before any mycorrhizal studies were made. More recently, some few local lists of exotic fungi in coniferous plantations have been published and, in addition, there are numerous references to the exotic nature of individual species. In regard to the fungal species, however, such references may be unreliable, since they have usually been made by foresters or other amateurs, whereas specialists on fungal taxonomy are needed for reliable specie determination.
Lists of mushrooms under exotic plantations have been published, for instance, by Birch (1937) and Rawlings (1950) for New Zealand, Purnell (1957) for Australia, Stephens and Kidd (1953a, b) for South Africa. According to these lists the most common and conspicuous mushrooms of the European forests occur now almost everywhere under exotic coniferous plantations, as Boletus bovinus, B. edulis, B. granulatus and B. luteus under pine, and B. elegans under larch. The common fly mushroom (Amanita muscaria) and some other Amanita species, Lactarius deliciosus and Hebeloma crustuliniforme are other typical associates of pines, as well as the gasteromycetes Rhyzopogon roseolus and R. luteolus and Scleroderma spp. Indigenous fungi of North America also grow under exotic Douglas fir plantations, as Boletus lakei in New Zealand (Rawlings, 1950) and B. amabilis in central Europe (Moser, 1967).
Mycorrhizal fungi of some mycorrhizal deciduous tree species have also followed their hosts to exotic plantations, for example, Amanita phalloides with oaks and Pisolithus tinctorius with eucalypts (Stephens and Kidd, 1953b; Reichert and Avizohar- Hershenzon, 1959).
SPECIFICITY AND ADAPTABILITY OF MYCORRHIZAL FUNGI
In general, mycorrhizal fungi are not exacting in regard to their host species. If a tree species is introduced to a new area without its ordinary mycorrhizal symbionts, it may first develop mycorrhizae with some indigenous fungi; later on, however, these are replaced by more specific symbionts, which are introduced intentionally or arrive incidentally. Apparently Boletus granulates, B. luteus, Lactarius deliciosus, and other indigenous fungi of the pine forests of Europe and North America are better adapted to pines and these fungi therefore predominate in exotic pine plantation throughout the world. It may also be presumed that these specific " pine fungi " are more efficient symbionts, that is, more beneficial to pines, than ubiquitous facultative species.
Although most known mycorrhizal fungi can infect a great number of tree species, there are others which are more specialized in regard to their hosts, for example, Boletus elegans (Suillus grevillei) on Larix, and Amanita phalloides on oaks.
Fortunately, Boletus granulatus and B. luteus, the most constant companions of pines in exotic plantations, are probably quite good symbionts. It is not sure, however, whether the same species are the best associates for spruce and. Douglas fir and other conifers as well. In fact, there are several examples to suggest quite the opposite (Pryor, 1958; Rawlings, 1958; Bryan, personal communication).
Different species of pine differ very little from each other with respect to their mycorrhizal relations. There may be, however, some differences in the susceptibility to mycorrhizal infection. To nurserymen of tropical countries, for instance, it is quite a familiar experience that seedlings of Pinus elliottii and P. caribaea turn yellow a few weeks after germination but soon resume a green colour and start to grow, whereas such a chlorotic phase seldom occurs in the development of P. radiata. This difference seems to be connected with the commencement of mycorrhizal infection. Mycorrhizal development starts earlier in Pinus radiata than in P. elliottii and P caribaea and therefore the former species has no chlorotic stage, whereas the recovery of the green colour in the latter species coincides with the development of the first mycorrhizae.
Many mycorrhizal fungi are very adaptable to widely different climatic conditions, e.g., Boletus granulatus. All fungi are not equally adaptable, however. Thus two of the commonest and most characteristic mycorrhizal fungi of the cool boreal forest, Boletus luteus and B. variegatus, are conspicuously different. The former grows almost everywhere in subtropical and even tropical pine plantations, whereas the latter can hardly be found anywhere outside its natural range. The different adaptability of different mycorrhizal species must be considered if species are selected for inoculation purposes.
The adaptability of fungal species to different soil conditions also deserves attention. Ectotrophic mycorrhizal fungi are usually acidophilic and, therefore, inoculation of alkaline soil does not succeed without concomitant acidification. There are, however, mycorrhizal fungi that grow naturally on calcareous soil. Boletus granulatus seems to be quite adaptable with respect to soil pH; at least, it is reported to be one of the dominant species both in the natural forests of Pinus halepensis on limestone rocks in Israel (Reichert and Avizohar-Hershenzon, 1959) and in plantations of the same species on alkaline soils in La Pampa Province in Argentina. These data are surprising because, in pure culture experiments, Boletus granulates has a rather narrow pH range with an optimum at 5.0 and does not grow at all at a pH above 7.0 (Models, 1941). Probably Boletus granulatus is a collective species including different subspecies and varieties or even several different species. As a whole, the taxonomy of the mycorrhizal fungi of exotic plantations deserves intensive study in which special emphasis should be placed on their physiological and ecological properties.
SOIL INOCULUM
If mycorrhizal inoculation is practiced at all, the soil of natural forests, plantations or nurseries is the most widely used form of inoculum. The techniques of applying soil inoculum vary according to nursery practice. If the seedlings are raised in beds of natural soil, the first inoculation is usually made by spreading a thin layer of mycorrhizal soil, 1 to 2 centimetres thick, on top of the bed and mixing it with the surface soil. No later inoculation is usually needed (Letourneux, 1957).
If the seedlings are raised on artificial soil in pots, polythene tubes, trays, or Swaziland beds, as is done in most tropical and subtropical countries, the inoculum (pine soil) is usually incorporated into the soil mixture used for potting or transplant beds. Thus, the mixture of May (1953), which has been recommended for East African nurseries, contains between 10 and 20 percent of pine soil.
The amount of pine soil was a problem in former times, when it was not readily available. Parry (1953), for instance, refers to the great difficulty of obtaining sufficient amounts of mycorrhizal soil when afforestation projects are started in new areas. Under such conditions it is advisable to use as small an amount as is still effective; 10 percent has been considered sufficient.
In the tropical nursery technique, seedlings are transplanted soon after germination, before they have any laterals or short roots, and inoculation of seedbeds would therefore be useless. Recently, however, some nurseries have cut out the transplanting of seedlings, and seed is sown directly into inoculated soil in polythene tubes.
Instead of forest topsoil, pine forest litter is used in some nurseries for mycorrhizal inoculation. Pine needle litter is also used as mulch in nurseries, for instance in the United States and Brazil, and has proved to be an efficient carrier of mycorrhizal infection. If the nursery soil has been sterilized with steam or methyl bromide, as is customary in the United States, unsterilized pine needle mulch greatly promotes the formation of mycorrhizae.
Instead of incorporating soil inoculum into the potting mixture, polythene tubes or bags are inoculated individually in some nurseries, either at the time of transplanting or a few weeks later.
Mycorrhizal soil or forest litter has been used in the Soviet Union to inoculate pine seed and acorns when shelterbelts have been established in the steppes by the direct seeding method (Lobanow, 1960). According to Krasnovskaya and Smirnova (1950), soaking of acorns in a water suspension of forest humus is sufficient for mycorrhization and is recommended in cases where the supply of large amounts of mycorrhizal soil and organic matter is difficult. The best infection and development of seedlings, however, are obtained if both mycorrhizal soil and organic manure are applied to the sowing patches. Lobanow (1960, pages 280-284) gives detailed instructions for the preparation, storage, and application of mycorrhizal soil.
Inoculation of pine seed with mycorrhizal soil has been tried in Australia (Forrest, 1966). Broadcasting the seed and mycorrhizal soil simultaneously has been successful in small-scale trials. The need for large amounts of soil inoculum (about 1 ton per acre or 2 1/2 tons per hectare), however, makes this method impracticable on a large scale. Experiments with seed pelleting, i.e. coating pine seed with mycorrhizal soil slurry in a suitable sticker, have not yet resulted in practicable application.
When mycorrhizal soil inoculum is transported for long distances, it is advisable to do this as rapidly as possible, keeping the soil moist. There are only a, few records of the drying of the inoculum and death of the fungi during transportation, but usually transportation has not taken more than two weeks. Probably failures have not been uncommon, however, since, according to Letourneux (1957), "nine times out of ten failure occurs if the shipment takes more than eight days. "
It is not known exactly how long mycorrhizal fungi can remain viable in soil under different storage conditions. According to Olatoye (1966), inoculum could be stored for 15 months without deterioration if it were kept moist in polythene bags. Other experiments have shown that some mycorrhizal fungi can survive, probably in the form of spores or sclerotia, for a long time even in dry soil. Storage of soil in a dry condition may, however, exert a considerable selective influence on its fungal population, some species being more drought. resistant than others.
Soil as a mycorrhizal inoculum has both advantages and disadvantages. The main advantages are the great ease and reliability with which inoculation can be carried out. It may be difficult to keep the fungi in viable condition during long transportation, but when the inoculum has once been introduced, it can be rapidly propagated and distributed. If the nursery is located close to existing plantations large amounts of inoculum can be used, which guarantees rapid and even infection of all the seedlings. If the inoculum comes from natural stands or healthy and thriving plantations, it presumably contains a balanced population of different species of mycorrhizal fungi-provided, of course, that no species have died during transportation. Mycorrhizal nursery soil, in turn, is a very homogeneous material for inoculating new beds or even new nurseries.
The bulkiness of soil needed for inoculation is perhaps the greatest technical drawback of the method. There are biological reasons, too, for which the use of soil inoculum has been strongly criticized by many scientists (Harley, 1959). Soil inoculum contains indiscriminately all the fungal species and other organisms of the site from where it is taken. It may also contain parasites and diseases. The danger of introducing diseases is rather slight if soil is brought from healthy natural stands to nurseries of the same area. Considerably more risk is involved in carrying soil from one nursery to another, when damping-off fungi and other nursery pests may be introduced at the same time. Import of soil from other countries always involves a risk of introduction of new diseases.
For this reason there has been great difficulty in bringing mycorrhizal inoculum to many countries. To protect against plant and animal diseases most countries have quarantine regulations, prohibiting or restricting the import of unsterilized soil and fresh plant material. In fact, the first introduction of mycorrhizal inocula to several areas has taken place illegally. This may be one reason why the introduction of mycorrhizal infection to many countries is very poorly recorded.
MYCORRHIZAL SEEDLINGS
Mycorrhizal seedlings were first used for inoculation of new nursery beds in Indonesia (Roeloffs, 1930). Since then the same technique has been regularly used there and is still used today (Becking, 1950; Alphen de Veer et al., 1954; Lamb, 1966; Cooling, 1967). It is therefore known as the Indonesian method, although the technique was known quite early in Australia as well (Kessell, 1927). The method is used in some other areas too, such as Nigeria, Madagascar and New Guinea.
According to the Indonesian method, mycorrhizal seed rings or " mother trees " are planted at 1-metre intervals in the nursery beds. The following year young seedlings, 6 to 8 weeks old, are planted around these mother trees at a spacing of 10 × 10 centimetres. A healthy green colour and start of growth are visible indications of the spread of the infection front the mother trees to the surrounding transplants. According to Becking and Alphen de Veer et al, the beds become sufficiently infected in two years, after which time mother trees are no longer necessary. The use of mother trees is said to be the only possible way to inoculate Pinus merkusii seedlings in the nursery; trials of mycorrhizal soil from pine plantations have failed.
As the original introduction of mycorrhizal infection to many countries has taken place in the roots of living seedlings, these can also be used for the preservation of mycorrhizal infection.
The use of mycorrhizal seedlings seems to be a very reliable method of inoculation; it is even reported to have succeeded in some cases when soil inoculum had failed (Indonesia, Madagascar). The sometimes slow advance of infection from the mother trees may be mentioned as a disadvantage.
Regarding the risk of introduction of diseases, mycorrhizal seedlings may be safer than soil inocula. The health of potted seedlings can be better controlled than large amounts of unsterilized soil.
PURE CULTURES
Theoretically, the use of pure cultures of mycorrhizal fungi would be by far the best method of inoculation. With pure culture inoculation the species of fungi could be chosen and all risk of introducing diseases would be eliminated. For successful use of pure cultures, however, it should be known which fungal species arc; the most beneficial symbionts and which are less efficient, how to culture the superior species to produce sufficient amounts of inoculum, and how to perform the inoculation in practical nursery or field conditions. In all these respects knowledge is still very deficient and, therefore, pure culture inoculation has been little practiced in forestry. On the other hand, pure cultures have been extensively used in many kinds of scientific experiments.
Data on the application of the pure culture technique on a field scale are available only from Austria and Argentina. The Austrian technique is mainly based on the extensive studies of Moser (1958a, b). The typical and probably most active mycorrhizal fungus of subalpine Pinus cembra forests, Boletus (Suillus plorans, is apparently absent in the nursery soils of the valleys and also in the alpine meadows, which were deforested centuries ago and should now be replanted (Göbl, 1965). The technique, therefore, aims at inoculation of Pinus cembra seedlings with Boletus plorans in the nursery. Since nursery soils may contain some other mycorrhiza-forming fungi, a heavy application of inoculum is necessary and a large amount of mycelium is needed. On the basis of the prolonged experiments of Moser (1958b) a routine technique has been developed.
Boletus plorans is first grown on Moser's (1958a, page 36) nutrient solution in Erlenmeyer flasks. From the flasks the mycelia are transferred to the same solution in 10-litre tanks, which are aerated for 2 to 3 hours daily. After 3 to 4 months, the liquid with mycelia is poured into 5-litre flasks with a sterilized mixture of vermiculite and shredded peat, and a sufficient amount of the same nutrient solution is added to moisten the substrate. Sufficient moisture and aeration are important for the growth of the fungus. Boletus plorans will grow throughout the substrate in a few months, after which the inoculum is ready for use.
The inoculum is sent from the laboratory to the nursery in polythene bags not more than three days before transplanting. The inoculum is applied to the beds at a rate of 3 to 4 litres per square metre and worked lightly into the surface soil, and seedlings are immediately planted in the inoculated bed. The success of the inoculation also depends on the physical and chemical properties of the nursery soil; in other words, it is not enough merely to introduce the fungus into the soil, but the conditions must be suitable for its further growth. Good aeration, moderate acidity, high organic matter content, and application of phosphorus fertilizers promote the growth and infectivity of Boletus plorans.
Inoculation is repeated, if possible, at every transplanting, regardless of whether the soil has been inoculated before or not. Apparently conditions are not favourable for the subalpine Boletus plorans in the nursery soils of valley bottoms and therefore the population must be strengthened from time to time. The greatest difficulty of the Austrian inoculation method is the very large amount of inoculum needed (3 litres per square metre corresponds to 30 cubic metres per hectare).
Inoculation is practiced in Argentina when new nurseries are established in formerly treeless areas, where mycorrhizal fungi are presumably lacking. The pure culture technique has been introduced by
Takacs (1961, 1964, 1967). The technique of preparation of the inoculum is rather similar to that of Moser. Fungi are first grown in liquid cultures and then transferred to 200-ml flasks containing peat or wheat-chaff substrate moistened with the same nutrient solution. After two months' incubation the inoculum is ready for distribution to the nursery. The stock cultures are maintained and the inocula raised in the mycorrhiza laboratory of the Instituto Nacional de Tecnología Agropecuaria (INTA) at Castelar.
Several species of fungi are used for inoculation. Because it is not known which one is preferable in different conditions, a mixture of three or four species is used for inoculation. Five flasks of each species, that is 15 or 20 flasks altogether, are sent from the laboratory to a new nursery. On arrival at the nursery the contents of each flask are mixed with 5 to 10 kilogrammes of sterilized soil or litter, which is then kept moist for three weeks before application to the nursery beds; 20 kilogrammes of such inoculated soil is sufficient to inoculate 80 to 100 square metres of nursery beds. Thus, with 20 flasks of pure culture inocula some 100 kilogrammes of soil inoculum can be prepared, which is enough for a nursery area of 500 square metres. Inoculation is performed in connexion with the preparation of nursery beds.
Practical inoculation with pure cultures is still young in both Austria and Argentina, hardly having progressed beyond the experimental stage.
Experiments in pure culture inoculation have also been conducted in several other countries. Lobanow (1960) lists several Soviet authors who have reported good effects of pure culture inoculation on oak seedlings; the technique of inoculation, however, is not described in detail. The Russian experiments have also been reviewed by Levisohn (1958). Successful inoculation of unsterilized soil with pure cultures on an experimental scale has been reported by Hatch (1936), Young (1940), Rayner and Levisohn (1941) and others. On the other hand, there are numerous experiences of less satisfactory or of inconsistent results. Scientists are more inclined to publish positive results of their experiments than failures, and therefore failures are probably much more frequent than would be concluded from the literature.
Pure culture inoculation is the only acceptable method for strictly scientific studies and is mainly used in experiments under aseptic conditions. The possibilities for practical field use, however, are still rather limited.
SPORES AND FRUITING BODIES
The spores of mycorrhizal fungi are hard or even impossible to germinate in laboratory conditions. On the other hand, in nature mycorrhizal fungi spread easily through spores (Robertson, 1954). Quite naturally, then, foresters have tried mycorrhizal inoculation by using spores or fruiting bodies of mycorrhizal fungi.
Thus, large amounts of Rhizopogon luteolus sporophores were used to inoculate nursery soils in Western Australia in the 1920s (Kessell and State, 1938) and a similar practice is said to be common in the Philippines (Lamb, 1966).
In all the above examples the technique has been the same-fresh sporophores have been collected and crushed and mixed with the surface soil of nursery beds. Dried sporophores have also been tried, although the results of these trials have not been published. It is known that dried sporophores of Boletus luteus were used in 1958 in two nurseries near the Kenyan east coast; the effect was probably positive, although not consistently so. Dried sporophores of Boletus luteus and Hebeloma crustuliniforme have also been sent from Kenya to the Sudan and Ghana for nursery inoculation; the outcome is, however, unknown (Gibson, personal communications).
Application of sporophores, or spore suspension, either alone or together with soil, has also been tried in the U.S.S.R. (Chastukhin, 1950; Klyushnik, 1952; Lobanow, 1960) and in Australia (Forrest, 1966).
Theoretically the method of using spores for inoculation has many advantages, especially for shipment of inoculum over long distances; the weight and size of the inoculum would be very small and the risk of introduction of diseases could be minimized. If the spores of mycorrhizal fungi remain alive and viable for a whole year even in dry conditions, as has been reported (Lobanow, 1960), the method would be really easy and attractive. For afforestation by direct seeding it would be an ideal solution. At present, however, too little is known about the germinability of the spores of different species and the factors affecting germination for spore or sporophore inoculation on a field scale to be recommended.
It is not known with absolute certainty whether there exist any natural forest or grassland soils which are completely lacking in fungal species capable of forming ectotrophic mycorrhizae with introduced pines or other ectotrophic trees. Although afforestation trials failed in numerous countries before the introduction of mycorrhizal inoculum, there still remains the possibility that some potential mycorrhiza-formers were present, but that they were either inefficient symbionts or in an inactive state. The natural spread of mycorrhizal fungi also seems to be very easy. It is interesting to observe that the most convincing evidence on the indispensability of mycorrhizal inoculation has usually come from remote islands, dike Fiji, Puerto Rico, and Trinidad, that is to say, from countries where the chances of spore flight or of accidental arrival of infection by other routes are minimal. Under such conditions artificial inoculation is most important.
On the other hand afforestation near existing natural forests or plantations of the same species may succeed without inoculation, as is shown by experiments with direct seeding in Australia (Forrest, 1965). Even then, however, inoculation may be justified because of faster mycorrhization and, consequently, better survival and initial growth of the seedlings.
For succesful inoculation, improvement of soil conditions is often essential. especially if the soil already contains :mycorrhizal fungi. Pure culture inoculation, in particular, requires special measures to guarantee the survival and thriving of the introduced fungi. Acidification and increase of organic matter are usually, the most beneficial measures. These measures alone, without inoculation, may some-times induce mycorrhizal development, indicating that mycorrhizal fungi were present, but that owing to adverse soil conditions they were in an inactive state (Rayner and Neilson-Jones, 1944). Several authors (Lobanow, 1960, pp. 255-259; Puschkinskaja and Mischustin, 1963) have expressed the opinion that in the forest steppe zone of the U.S.S.R. mycorrhizal inoculation is unnecessary, because good growth and mycorrhization of pine and oak seedlings can be achieved by organic or inorganic fertilization and improvement of soil moisture conditions. On the other hand, studies conducted in the prairie forest belt of the United States (Wilde, 1954) strongly support the necessity of mycorrhizal inoculation in that area.
In addition to organic matter and acidification, phosphorus fertilization seems to be one of the most effective measures for activating mycorrhiza-forming fungi (Me Comb and Griffith, 1!346).
Several authors have emphasized the importance of early mycorrhization in dry areas (:Parry, 1953, 1956). Mycorrhizal association, however, is in all probability indispensable for mycotrophic trees in areas with moist climates as well. Therefore, the question arises why the most serious difficulties due to mycorrhizal deficiency have always been experienced in areas with a pronounced dry season. The following reasons may be suggested:
1. The greatest advantage of the ectotrophic mycorrhizal association usually lies in increasing the water uptake and drought resistance of the trees. Thus if seedlings, once planted out, are not mycorrhizal by the commencement of the first dry season, they cannot survive. On the other hand, in moister climates tile seedlings can survive longer without mycrrohizae and, therefore, have better chances to become infected by either airborne spores or indigenous soil fungi.2. The indigenous soil population of mycorrhizal fungi, where it exists, is perhaps denser in moist climates. The longer the dry season, the greater is the probability of the mycelia dying out in the soil, whereas a continuously moist soil with a reasonably high content of organic matter is a favourable environment for survival.
3. Moist soils offer better substrates for the germination of fungal spores and, consequently, greater chances for airborne infection than dry soils. A humid climate also promotes the production of fungal sporophores and spores.
For all the above reasons mycorrhizal inoculation or other measures to secure good early development of mycorrhizae are particularly important in dry areas. The ecological significance of ectotrophic mycotrophy, however, has also been emphasized in cold climates. Because the climates near alpine and arctic timber lines are humid without any pronounced dry season, there must be other factors besides moisture that favour the mycotrophic mode of nutrition. Moser (1967) has suggested that mycotrophy enables the trees to use the short growing season more effectively to obtain a sufficient amount of nutrients for the long cold season and attain the necessary resistance before the early frosts. Probably the advantage of mycotrophy in cold climates is also connected with the nitrogen nutrition of the trees. Mycorrhizal fungi have a greater ability than their hosts to utilize complex organic nitrogen compounds and, therefore, since nitrogen mobilization is extremely slow in cold arctic and alpine soils, trees can better fulfil their nitrogen requirements in symbiosis with fungi.
The degree of mycotrophy is somewhat variable among mycotrophic trees. Although probably all of them are unable to grow without fungal association under natural forest conditions, some species seem to be more exacting than others in regard to specific fungal symbionts, and there are also differences in the ease with which they become infected. Of tropical pines, according to Asian experience (Letourneux, 1957), Pinus merkusii, P. khasya, and P. insularis are the most dependent on early mycorrhization. P. radiata and P. patula are usually infected more rapidly than P. caribaea and P. elliottii. Probably some other conifers are more specific than pines, for example Douglas fir, spruce and larch (Bergemann, 1955; Pryor, 1958; Rawlings, 1958).
On the other hand, other ectotrophic trees are less dependent on fungal symbiosis than pines; Lobanow (1960) calls them weakly mycotrophic. There may exist a whole range of trees from nonmycotrophic species through weakly mycotrophic ones to obligate mycotrophy. From the point of view of exotic plantations, Eucalyptus species deserve attention. In general, no mycorrhizal difficulties have been experienced when eucalypts have been introduced into new areas and they have therefore been supposed to be independent of the presence of mycorrhizal fungi (Letourneux, 1957). Latterly, however, ectotrophic mycorrhizae have been found on practically all species of Eucalyptus. Mycorrhizal symbionts of Australian eucalypts have followed the hosts to new areas, and the indispensability of mycorrhizal inoculation for one group of eucalypts, Renantherae, has been suggested (Pryor, 1956a, b).
Practical experience has shown inoculation to be unnecessary for trees with endotrophic mycorrhiza. African nursery instructions, for instance, particularly emphasize that soil inoculation is not necessary for Cupressus lusitanica. Cypress plantations have been successful in many countries, where at the same time pine trials succumbed because of mycorrhizal deficiency. The obvious reason must be the presence of appropriate endotrophic fungi practically everywhere. Endogone species, the probable symbionts of vesicular-arbuscular endotrophic mycorrhizae, seem to be quite cosmopolitan in regard to both geography and host species.
Mycorrhizal fungi can survive for a long time in soil even without tree vegetation. There is therefore usually no need for inoculation of new nurseries on former agricultural soils if the area was once forested with ectotrophic trees. With prolonged agricultural use, however, especially if lime has been applied, some impoverishment or undesirable selection of the mycorrhizal population may take place and inoculation is recommended for soils which have long been under agriculture (Wakeley, 1954; Stoeckeler and Jones, 1957).
Soil sterilization is a common practice in modern nursery management and its influence on mycorrhizal fungi deserves particular attention. Fungicides usually delay the commencement of mycorrhizal infection but do not prevent it entirely. The lag varies, depending on both the type of fungicide and the strength of application. In nurseries where soil sterilization is a regular practice, the delay of mycorrhizal infection may be disastrous if the seedlings are still nonmycorrhizal at the time of lifting, such seedlings having a low survival in the field. The development of root systems :must therefore be repeatedly inspected in the nurseries, and mycorrhizal inoculation should be performed if desirable. Mycorrhizal development also deserves special consideration in the grading of nursery stock. The use of unsterilized pine needle litter as mulch, customary in many southern nurseries in the United States, is based on sound reasoning, the mulch functioning at the same time as mycorrhizal inoculum.
Soil sterilization can also improve the conditions for mycorrhizal infection Sterilization reduces competition and, if a suitable source of infection is present, mycorrhizal development may be much faster than in unsterilized soil.
The selection of mycorrhizal fungi for different climatic and soil conditions has so far received little attention. Until more is known about the ecological properties of individual fungal species, the most reliable method is to procure inoculum from conditions which correspond as nearly as possible to the conditions of the area where the inoculum will be used. Of individual species, Cenococcum graniforme is known to be especially drought resistant (Worley and Hackskaylo, l 959; Trappe, 1964) and might be used for inoculation in particularly dry conditions. Mycorrhizal fungi also have very variable temperature requirements. Sensitivity to high temperatures may be the reason for the absence of Boletus variegatus in subtropical pine plantations. Pisolithus tinctorius is probably one of the most suitable species for tropical conditions. It has relatively high temperature requirements (Marx, 1966) and also grows naturally in the tropics; in the temper ate zones it is often found growing on extremely -warm sites, such as the black waste banks of coal mines (Schramm, 1966). Thelephora terrestris is another species that is adaptable to warm climates; its symbiotic efficiency, however, is so far unknown. Pisolithus tinctorius and Telephora terrestris have often been the first fungi whose sporophores have appeared in exotic pine plantations in tropical countries. Selection of suitable mycorrhizal fungi may also be of great importance in the case of the afforestation of exceptionally cold areas, at and above the timber line in the mountains (Moser 1956, 1962).
Ectotrophic mycorrhizal fungi are usually acidophilous, and acidification of alkaline soil is often an indispensable requisite for successful inoculation or an effective measure to stimulate existing fungi. There are numerous ectotrophic tree species (such as Pinus nigra and P. halepensis, Quercus spp.), however, which prefer an alkaline substrate and form forests even on limestone rocks. In all probability these forests must contain a natural population of basophilous mycorrhizal fungi, which should be used to inoculate alkaline soils to be afforested.
FOREST RESEARCH
The application of mycorrhizal inoculation in forestry practice is based mainly on hypotheses and field experience, and relatively little is known with certainty. In the face of their tremendous afforestation programmes, developing countries are in urgent need of research in order to obtain a, reliable scientific foundation for practical work and to guarantee its successful outcome. Some important subjects for mycorrhizal research in the immediate future are:
1. Comparative studies on the physiology, ecology, and symbiotic efficiency of different fungal species. It would be most desirable to find suitable mycorrhizal fungi for exceptional conditions, such as alkaline or extremely acid soils, permanently or temporarily waterlogged soils, dry or hot climates, etc.2. Development of a practicable technique of pure culture inoculation. With a pure culture technique it would be possible to encourage the use of the most efficient species while avoiding the risk of introducing pests.
3. Developments of a technique of seed inoculation.
4. Further studies on mycorrhizal relations among the poorly known ectotrophic trees. The most important of these are members of the genera Eucalyptus and Nothofagus, and the group also includes other tropical and subtropical species of Fagaceae, and the families of Dipterocarpaceae and Cesalpiniaceae.
5. Endotrophic mycorrhizae of forest trees. This represents a more intimate form of symbiosis and a greater diversity of physiological relationships than the relatively uniform ectotrophic mycorrhizae (Mosse, 1963).
6. Geographic distribution and migration history of exotic mycorrhizal fungi. Although this field of study may not be of any great practical importance to afforestation, ecologically it is a problem of primary interest and is recommended particularly for the botany departments of universities in countries with considerable exotic plantations.
FIGURE 3. - Inoculation individual seedlings in polythene tubes. Bukuru nursery, Nigeria.
FORESTRY PRACTICE
In forest countries where ectotrophic trees and fungi are indigenous, there is little need or possibility of mycorrhizal inoculation. If troubles connected with poor development of mycorrhizae appear in a nursery, a remedy is more likely to be found by improving the soil conditions (acidity, fertility, aeration, content of organic matter, etc.) than by the introduction of new fungi to the soil. Cases when inoculation is beneficial and practicable, as in the afforestation of alpine areas in Austria, are so far exceptions.
In areas where exotic trees have been grown for a considerable time and soils are well infected with mycorrhizal fungi-for example parts of Australia, south and east Africa, Latin America, and :New Zealand - the situation is almost the same. There, mycorrhizal inoculation is usually unnecessary. Where the density of mycorrhizal populations in the soils is low, at least locally, some safety measures may be advisable to guarantee early mycorrhization of the seedlings.
Soil is usually brought to tropical and subtropical nurseries from outside, for instance from natural forests, plantations or grasslands. The common practice of adding between 10 and 20 percent of pine soil to the soil mixture for pots, tubes, trays or Swaziland beds is not necessary in old nurseries, which are already well infected with mycorrhizal fungi; however, it does no harm. This technique was adopted at a time when pine soil was not easily available and soil of natural broad-leaved forests or grasslands was used with the addition of as small an amount of pine soil as was effective. Today, when nurseries are often located in the midst of pine plantations, it is most practicable to bring all the soil needed in the nursery from these plantations. A moderate nutrient level is also necessary for good mycorrhizae; mycorrhizal development is often best promoted by phosphorus fertilization.
Today, soil for seedbeds and transplant pots may be sterilized in tropical and subtropical nurseries. Although even then inoculation has usually proved unnecessary in old nurseries, some kind guarantee of mycorrhization is advisable. Early mycorrhization is promoted with unsterilized pine litter mulch or by soil inoculation. Inoculation is performed by means of a small amount of unsterilized. mycorrhizal soil, which is thoroughly mixed with the sterilized soil before potting. If the soil is reasonably acid and rich in organic matter, there is little risk of damping-off or other diseases.
The inoculation of new nurseries is recommended, although they may also become infected naturally. Artificial inoculation assures a more rapid and even infection of the whole nursery. Soil inoculum may be used if it can be obtained locally from a healthy and vigorous plantation. For longer distances, richly mycorrhizal potted plants are preferable. Eucalyptus, cypress, and many other species do not need any inoculation.
For the importation of mycorrhizal inoculum from abroad, pure cultures are least subject to risk (Bakshi, 1967), and the same method is also advisable for long-distance transfers inside countries. But the technique is as yet poorly mastered. Regarding the most efficient fungal species, very little is known with certainty. In all probability, Boletus granulatus is an efficient species for pines, perhaps the most efficient one, and very adaptable to widely different ecological conditions. At the present stage of our knowledge, however, it is not advisable to use one single species for inoculation. Other species which may be recommended for use together with Boletus granulatus are B. Iuteus and B. edulis, Rhizopogon roseolus and R. luteolus, Amanita muscaria and Lactarius deliciosus. For alkaline soils, a mixture of Mediterranean Boleti may be preferable.
If, however, pure culture inoculation fails, then importation of soil or living seedlings is necessary. Soil should be taken from conditions as similar as possible to those of the area to be afforested. To reduce the risk of introducing diseases, healthy natural forests should be preferred and only the necessary amount of soil should be brought in. Soil can also be treated with some insecticide which does not damage fungi.
PLANT QUARANTINE
Most countries have strict plant protection regulations which prohibit the import of unsterilized soil and require a certain quarantine period for imported living plants. These regulations usually ignore mycorrhizal inoculation and may, in fact, make the legal import of mycorrhizal inocula impossible. Special regulations for the international exchange of mycorrhizal material are needed.
An essential requirement for the successful importation of mycorrhizal inoculum is that delivery of the material (soil, living seedlings, or pure cultures of fungi) can be effected rapidly without prolonged delay at quarantine stations. In fact, the need for import of soil or diving seedlings for mycorrhizal inoculation is relatively restricted. and exists only at the first introduction trials of pines and some other conifers to countries where pines have not previously been grown. For further inoculation, mycorrhizal material can then be obtained locally.
There is a good case for a set of internationally agreed regulations to govern the exchange of mycorrhizal inocula and for an information service as to the sources of inoculum available. This would be an appropriate field for action by IUFRO, FAO, the Organization for Economic Cooperation and Development (OECD) and other international organizations.
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