Improving Propagation Success of Dalbergia Melanoxylon (African Blackwood) in Tanzania (I): Characterization of Mycorrhiza Associated with D

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IMPROVING PROPAGATION SUCCESS OF DALBERGIA MELANOXYLON (AFRICAN BLACKWOOD) IN TANZANIA (I): CHARACTERIZATION OF MYCORRHIZA ASSOCIATED WITH D. MELANOXYLON (AFRICAN BLACKWOOD) IN TANZANIA

1WBA Washa, AMS Nyomora2 and HVM Lyaruu2 1Mkwawa University College of Education, P.O. Box. Private Bag Iringa, Tanzania 2University of Dar es Salaam, P.O. Box 35060 DSM, Tanzania Corresponding author: E-mail: [email protected]

ABSTRACT Dalbergia melanoxylon Guill & Perr is a plant with quality wood in the World and therefore is over harvested for timber while its regeneration capacity is very low. The propagation techniques such as tissue culture or use of mycorrhiza have not been investigated which instigated conduction of this study by investigating the presence of mycorrhiza that might be useful for its regeneration. Some 120kg of soil and 120 roots of D. melanoxylon from Kilwa, Kilosa and Babati were sampled to identify mycorrhiza related with regeneration of the species. Cleaning the tissues was done by soaking the root pieces in 1.79M KOH, and 0.1M Hcl. Staining was done using 0.05% Trypan blue and de-staining in 14:1:1 lactic acid: glycerol: water and mounting on slides for observation. Soil was soaked in water overnight and the mycorrhiza was separated using a stereo microscope at 50X. Separated mycorrhiza were incubated for observation using a compound microscope at 400X. Twenty six (26) ECM resembling Inocybe and Laccaria sp were isolated from the root pieces and 18 VAM resembling Glomus species were isolated from soil. It is recommended that future research should investigate proper inoculum types and time for inoculating the species in nurseries for propagation.

Key words: Dalbergia melanoxylon, Ectomycorrhiza, Endomycorrhiza, Mycorrhiza

INTRODUCTION plant roots are metabolized by plants leading Mycorrhiza is a symbiotic association to improved plant growth including between a fungus and roots of a vascular regeneration (Munyanziza et al. 1994). plant. In mycorrhizal association, the fungus Plants having mycorrhizal associations are colonizes the host plant's roots, either often more competitive and more resilient intracellularly as in arbuscular mycorrhizal and tolerant to environmental stresses fungi (VAM), or extracellularly as in including diseases, pathogens and drought ectomycorrhizal fungi (ECM). They are an compared to plants that do not have such important component of soil life and soil association. Ectomycorrhiza have been chemistry (Harley and Smith 1983, Jackson reported to produce Plant Growth Regulators and Mason 1984, Kanyagha 2008). The (PGRs) including IAA, ethylene and relationships are known to affect the bi- Pyrrolizidine Alkaloids (PAs) in their directional movement of moisture and association with plants which play a nutrients whereby carbon assimilated by the regulatory role between fungi and roots plant flows to the fungus and inorganic (Smith and Read 1997). These regulators are nutrients absorbed by fungus move into the yet to be verified in Dalbergia melanoxylon plant. In this way, a critical linkage between (Smith and Read 1997). the plant root and soil is formed. Nutrients delivered by the mycorrhiza fungi to the

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Classification of mycorrhiza includes and furniture while its natural regeneration Ectomycorrhiza (ECM), Endomycorrhiza is low and attains a harvestable age at 70 to (Vascular arbuscular mycorrhiza (VAM), 100 years. Orchid mycorrhiza, Ericoid and Arbutoid mycorrhiza (Brundrett et al. 1996). ECM or Dalbergia melanoxylon is wide spread in sheathing mycorrhiza belongs to the classes tropical Africa from Senegal and Cote Basidiomycetes and Ascomycetes that d’Ivore in the West, to Kenya and Ethiopia dominate temperate countries. These are in the East, and extending South to South characterized by having sheath of hyphae Africa. It is found in at least 26 sub-Saharan (mantle) around the roots to form network of countries (Nshubemuki 1993). In Tanzania, hyphae termed hartig net. Ectomycorrhiza Dalbergia melanoxylon is distributed in T4, are typically formed between woody plant T5, T6, T8 floristic regions which are roots. VAM belong to class Zygomycetes Shinyanga and Mwanza (T4), Itigi and which are known to dominate the miombo Dodoma (T5), Muheza and Matombo (T6) woodlands and are characterized by having and Mtwara (T8) (Redhead and Temu 1981). branching mycelia through the soil The species is not abundant in all regions connected to the roots of host plant forming where it is found and inhabit rainforest and arbuscules. ECM can also form vesicles. open miombo woodlands of Tanzania in Orchid mycorrhizae belong to class which rainfall is often marginal and drought Basidiomycetes that dominate many parts of periods are usual. Such areas are the World in orchidaceous plants and are environmentally fragile and their vitality characterized by forming intercellular coils depends on a delicate balance between or hypha aggregates within their host tissues. populations within them. Dalbergia Ericoid mycorrhizae belong to order Ericales melanoxylon has adapted well to this that are found mostly in the northern and marginal existence and its long growing southern hemisphere and are characterized period is an out come of this adaptation to by septate fungal hyphae which harsh conditions (Arbonnier 2004). The intracellularlly penetrate the host plant root. adaptation also covers the way it regenerates Arbutoid mycorrhizae (Ectondomycorrhiza) mostly by forming sapling regeneration in belong to the genera Arbutus and are similar areas where roots gets wounded as it winds to ectomycorrhiza by forming sheaths. through rough rocky soil (Arbonnier 2004). Regeneration and adaptive ability of D. Plant preference for mycorrhiza seem to melanoxylon is thought to be contributed by follow both taxonomic and ecological line, mycorrhizal associated with it. for example, all Pine plants family is associated with Ectomycorrhiza, while the The low regeneration ability is contributed Rose plants family is associated with VAM. by low seed viability and seed germination Ericales are associated with ECM while and slow growth rate (Amri 2008, Washa VAM and most of tropical trees are and Nyomora 2011). Dalbergia melanoxylon associated with both VAM and ECM is classified as Lower Risk / near threatened (Munyanziza et al. 1994). in the IUCN red listing that is, it is neither endangered nor of least concern but nearly Dalbergia melanoxylon Guill. & Perr. is a threatened if propagation efforts on the flowering plant native to the seasonally dry species not are taken immediately. The regions of Africa (IUCN 2008). This species propagation of the D. melanoxylon species has one of the most valuable timber in the by advanced techniques such as tissue World and therefore is over harvested for culture and use of mycorrhiza fungi have not wood, carvings, timber, music instruments been investigated as reported by (Readhead

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and Temu 1981) which instigated Soaked root pieces were then stained in conduction of this study. The main objective acidic glycerol solution containing 0.05% of this research was to investigate the Trypan blue for 30 min while incubated in a presence of VAM and ECM in Dalbergia water bath at 90oC for contrast. The root melanoxylon found in Tanzania, identify pieces were de-stained overnight in 14:1:1 them and establish how they can be lactic acid: glycerol: water to remove stains employed in improving the species of other cells and non mycorrhizal tissues propagation techniques. and for more contrast (Kormanik and Mc Graw 1982). De-stained roots were mounted MATERIAL AND METHODS on slides for observation. Mycorrhizas were Isolation of mycorrhiza fungi (VAM and observed using a compound microscope; ECM) model Olympus CH30 RF200 at 400X. Soil samples from soil rhizosphere and short Features of the spores were usually used to root terminals were collected from identify Glomalean types in accordance with Dalbergia melanoxylon in Kilwa, Kilosa and (Kormanik and Mc Graw (1982). Babati Districts of Tanzania using standard methods as described by (Brundrett et Identification of ECM al1996). The criteria used to select the three Soil samples were soaked in water sites were prevalence of dense populations overnight, and then were cleaned by passing of Dalbergia melanoxylon, habitat them through gently running water. heterogeneity and their different Mycorrhizas were separated from their geographical locations in Tanzania. A total mantle under water in Petri dish using a of 120kg of soil samples were collected stereo microscope at x 50 of XSZ – 3G. within 5mm from their associated roots of Separated mycorrhiza were incubated in the selected sampling trees from which 20kg both lactophenol cotton blue and toluidine of the sampled soil were used for isolation blue dyes for 10 to 15 seconds for contrast to and identification of mycorrhiza. A total of ease microscopic observation. About 5gms 120 root terminals were extracted from the each of soaked soil were used on slides for top soil layer (approx. 15 cm) of a tree soil- this microscopic observation. Identity of the root zone by tracing them from the point of EMF was determined based on published attachment on the stem before severing them descriptions of different mycorrhiza types by from their stems using a Machete. A total of (Brundrett et al 1996) by comparing 20 root terminals of the collected roots were appearance of the mantle surface, associated used for isolation and identification of emanating hyphae and sclerotia. mycorrhiza. The samples were enclosed in polythene bags and transported in cool RESULTS boxes at 10oC to the Botany Department, Mycorrhiza fungi associated with D. University of Dar es Salaam for assessment. melanoxylon In total three species of mycorhiza fungi Identification of VAM were found associated with D. melanoxylon. Short terminal roots of D. melanoxylon from Two (ECM) mycorrhiza species Inocybe sp each site were cut into 1cm pieces. The and Laccaria species as shown in Plates 1 pieces were cleared in 1.79M KOH for and 2 respectively were identified. A total of 10min, maintained in water bath at 90oC for 26 mycorrhizae isolated from 20kg of soil 30 min, rinsed three times in distilled water rhizosphere based on the general appearance before soaking in 0.1M hydrochloric acid for and characteristics of dark brown colour, 2 hours to soften and acidify the tissues for fairly straight, slender, infrequent branching easing penetration of staining reagents. with hyphae emanating on the mantle

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surface and abundant clamp connections, while 18 mycorrhiza isolated from 20 root resembled only 2 species of Ectomycorrhiza pieces resembled Glomus

Plate 1 Plate 2

General characteristics observed for short branches, loose straggly hyphae which Inocybe sp were frequently close to the mantle surface Mycorrhizas were short and stubby, with without strands and sclerotia. Mantle edges frequently irregular branching pattern. The were loosely formed becoming compacted in loose fraggly hyphae were occasionally the older mycorrhiza. Emanating hyphae had found around the base of mycorrhiza. abundant large irregularly formed clamp- Detailed features observed for Inocybe sp connections and elbow-like protrusions. indicated that mycorrhizas were brown, These features were confirmed in (Bound.) (dark red brown), without strands and Pat. ITE for Laccaria proxima. In this sclerotia. Emanating hyphae had abundant particular study, mycorrhizae mantle looked clamp connections and elbow-like larger due to the presence of loosely cotton protrusions. These features were confirmed hyphae surroundings unlike that identified in in (Fr: Fr) Gillet. ITE as Inocybe petiginosa. the ITE that looked slender due to the In this particular study, Inocybe sp mantle absence of loosely cotton hyphae. Laccaria looked larger since it was surrounded by proxima is found in the Class loosely cotton hyphae while that identified Basidiomycotina, Order Agaricales and the in the ITE were slender and not surrounded family Tricholomataceae and it is commonly by loosely cotton hyphae.. Inocybe sp is associated with Pinus sylvestris, Picea found in the Class Basidiomycotina, Order sitchensis and Betula pendula. Agaricales and family Cortinariaceae. Inocybe petiginosa is commonly associated Glomus sp, the VAM associated with D. with Picea sitchensis and Betula pendula melanoxylon found mostly in higher altitudes and The identified VAM are as shown in plates temperate regions. 3-5. Mycorrhiza spores identified in plate 3 resembled Glomus sp in general appearance General characteristics observed for and characteristics. Laccaria sp Mycorrhiza fungi were fairly long and sinuous, with frequent irregularly spaced

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Plate 3: Plate 4:

Plate 5:

Hyphae were non septate and ramified Glomaceae and Genus Glomus (Sedaghati within the cortical cells. Arbuscules were 2002) and is frequently found in association intricately branched haustoria in cortical with sugar cane, grape and some grains such cells. Detailed features for the identified as wheat, barley, maize and sorghum Glomus sp indicated that a species had (Sedaghati 2002 relatively straight hyphae that ramified along the root cortex and often producing “H” DISCUSSION branches which resulted in simultaneous Like other woody and tropical trees which growth in two directions. These hyphae “H” were found associated with mycorrhizal usually stained relatively dark. Arbuscules fungi (Ingleby et al 1990; Kanyagha 2008), were dense and compact, had intracellularly mycorrhiza fungi has been found in D. developed mantle with cell to cell hyphae. melanoxylon for the first time in this study These features, spores and other information where it was confirmed that their presence found confirmed for Glomus versiforme was responsible for keeping D. melanoxylon (http://invam.caf.wvu.edu/fungi/taxonomy/s adaptive to a wide range of environments pecies.ID.htm) and in the reference including natural regeneration through accession IT104 in accordance with (Daniels saplings from root suckers. The high and Trappe 1979). Glomus versiforme is environmental adaptive ability of D. found in the Phylum Glomeromycota, Class melanoxylon seem to be contributed by its Glomeremycetes; Order Glomerales, Family association with the two ectomycorrhiza

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species, Inocybe and Laccaria and one penetrating their host roots forming hyphae endomycorrhiza species, Glomus. These network connections and protrusions that mycorrhizal associations are involved in the increases the surface area for the improved formation of high hyphae network moisture and nutrients acquisition. Possibly connections in the roots which facilitates inoculating Glomus in the nursery and absorption of a range of nutrients from the transplanted D. melanoxylon may increase soil and increasing penetration ability of the the adaptive ability of the species for roots into the soil (Borowics 2001). successful propagation. Glomus is an obligate biotroph, meaning that it requires a Inocybe petiginosa and Laccaria proxima living photoautotropic host to complete its have been reported to have a broad host life cycle and produce the next generation of range and an extensive geographical spores. Glomus species are also entirely distribution worldwide (Mason et al. 1987). asexual (Modjo et al. 1986). Glomus begins This is in agreement with the current as a spore produced outside or inside the findings since the two ectomycorrhiza host root, are able to germinate without a species were identified in Kilwa, Kilosa and host plant. If the spore is not already in the Babati which are found in different root, a germination tube is formed which ecological zones of Tanzania. The efficiency grows through the soil until it finds a host of the two ectomycorrhiza species in root. Glomus penetrates the roots and grows creating functional symbiotic relation to the between root cells or penetrates the cell wall host plants is indicated by the adaptive and grows within root cells to form ability and abiotic stress tolerance of D. arbuscules. The arbuscules are tree-shaped melanoxylon. Sexual reproduction which subcellular structures that form and connect maintains availability of their spores ensures plants to the hyphal network of the fungi their continuous presence in the soil and (Modjo et al 1986, Sylvia 2000, Kanyagha hence a sustained symbiotic relation 2008). Arbuscules are the main site for throughout the life of the host plant. nutrient exchange between Glomus and its symbiotic plant partner. Soil may contain Glomus exists in the environment both as over 100 meters of hyphae per cubic spore and hyphae which can form dense centimeter (Sylvia 2000). This network of network called mycelia, though most of hyphae is designed to increase the plants Glomus biomass occurs within the roots of uptake of important nutrients such as host plants. Glomus is believed to exist in all phosphates and water. In exchange for terrestrial habitats colonized by vascular nutrients and water, plants supply Glomus plants and may form an endosymbiotic with carbohydrates it needs to survive relationship with 70-90% of extant vascular (Sylvia 2000). plants. The Glomus-plant symbiosis plays an important role in the economic sectors Host specificity in VAM has led to link a involving plants growth (Modjo et al 1986) positive correlation in VAM diversity and so inoculating the media with Glomus plant diversity. For example VAM have during propagation of D. melanoxylon as been reported to be associated with Rose obtained from the results of this study may family plants, Ericales plant and most of speed growth and regeneration of the tropical trees (Munyanziza et al. 1994). species. The two ectomycorrhiza species Inocybe Glomus sp are successful in producing petiginosa and Laccaria proxima found spores in the soil around their host plant root associated with D. melanoxylon are the main that become a source of association by source of spore in the soil rhizosphere

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around D. melanoxylon roots. They are well melanoxylon seedling growth rate known to mobilize and convert nutrients into improvement. their available form for plant absorption ACKNOWLEDGEMENT (Sylvia 2000) hence probably they are The financial assistance from Mkwawa related to the high adaptability of University College of Education (MUCE) to D.melanoxylon in a wide range of habitat the PhD candidate Washa Bugalama Washa including the poor soil environments like is highly acknowledged. those in Kilwa, Babati and Kilosa. The EMF spores found in the soil rhizosphere are also REFERENCES known to forms hyphae network connection Borowics VA. 2001. “Do arbuscular to the host plant (Sylvia 2000). This may mycorrhizal fungi alter-plant pathogens also on the other hand enable a directional relation?” Ecology. 82(11): 3057-3068. flow of materials from the fungi to D. Brundrett M; Boughr N; Dell B; Grove T melanoxylon roots and vice-versa which and Malajezuk N. 1996. “Working with contributes to its success. mycorrhizae in forest and agriculture”. Australia Center for International CONCLUSION AND Agriculture Research. ACIAR Monograph RECOMMENDATIONS 32: 374pp. It is now evident thatDalbergia Daniels B A and Trappe J M 1979. Glomus melanoxylon form mycorrhizal association epigaeus sp. nov., a useful fungus for and the presence of mycorrhiza fungi in vesicular-arbuscular mycorrhizal roots and soil rhizosphere of the species are research. Canadian Journal of Botany responsible for the relatively higher adaptive 57:539-542. ability of D. melanoxylon to a wide range of Harley J L and Smith S E. 1983. habitat. Propagation of D. melanoxylon in “Mycorrhizal Symbiosis”, Academic new areas using the two ectomycorrhiza Press. New York 483 Pp species and the Glomus species can possibly Ingleby K; Mason PA; Las T and Fleming be accomplished by introducing the fungus LV 1990. “Identification of in the new areas using soil rhizosphere ectomycorrhizas”. ITE Research around D. melanoxylon roots mixed with D. Publication no. 5. London UK: melanoxylon root fragments to improve HMSO.112pp natural regeneration. However the proper Jackson R M and Mason P A 1984. time for introducing mycorrhiza in new D. “Mycorrhizae, studies in Biology no. 159” melanoxylon plantations is another area Edward Alrnold (publishers) Ltd. London which needs investigation. Mycorrhiza could pp 110-117 possibly also be used to acclimatize D. Kanyagha H 2008. Vegetative Propagation, melanoxylon propagules from Types and effects of Mycorrhizae on some micropropagation using an appropriate trees and shrubs endemic to Pugu forest protocol, again this needs further applied reserve, Tanzania. MSc thesis, University research. of Dar es Salaam. Kormanik PP and Mc Graw A 1982. It is recommended to future researchers to Quantification of VAM in plant roots. In investigate the mycorrhiza population and methods and principles of mycorrhizal the genetic diversity of Glomus, Inocybe and Research. Ed. NC. Schenck pp37-46. The Laccaria in D. melanoxylon, their American Pytopathological Society. multiplication and integration into the soil Lamrood P Y and Vaidya J G 2001. Fungal media, proper inoculums type and the genera of the Pune City. Department of appropriate time of inoculation for D. Botany, Pune University, India.

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