Mass Production of Arbuscular Mycorrhiza Fungi (AM Fungi) | (VAM Fungi)

The mass production or commercial production of VAM fungi (Vesicular Arbascular Mycorrhiza) or AM Fungi (Arbuscular Mycorrhiza Fungi) is given here.

1. Professor Jayashankar Telangana State Agricultural University College of Agriculture, Rajendranagar , Hyderabad-30 Submitted By : Arunodaya Maji CARA-2018-110 Batch - D Under the guidance of, Course In-charge : Dr. S. Triveni Mass Production of AM Fungi

2. CONTENTS : Introduction Types of mycorrhizae Mycorrhizal biofertilizer Applications of mycorrhizae Conclusion References

3. Introduction : Mycorrhizae are mutualistic symbiotic associations formed between the roots of higher plants and fungi. It is an Greek word, mykes : mushroom or fungi; rhiza : root. Fungal roots were discovered by the German botanist A B Frank in the last century (1855) in forest trees such as pine. In nature approximately 90% of plants are infected with mycorrhizae. 83% Dicots,79% Monocots and 100% Gymnosperms. Convert insoluble form of phosphorous in soil into soluble form.

4. An arbuscule of Glomus versiforme in a root cortex cell with branch hyphae densely packed in the cortex cell of the host Arbuscule of Gigaspora margarita with an elongated trunk hypha (T) and tufts of fine branch hyphae (arrows)

5. Developing arbuscule of Glomus mosseae in a root cell with fine branch hyphae (arrows). The trunk (T) of this arbuscule branched from an intercellular hyphae.

6. Mycorrhiza The term “mycorrhiza” was coined by A. B. Frank, 1885. Mutualistic symbiosis (non-pathogenic association) between soil-borne fungi and roots of plants. Key components of ecosystems Link plants within a habitat Labelled CO 2 fed to tree can be found in seedlings growing nearby Retain and conserve mineral nutrients

7. Types of mycorrhizae : On the basis of morphological and anatomical features, mycorrhizae are divided into the three types. 1. Endomycorrhizae 2. Ectomycorrhizae 3. Ectendomycorrhizae

8. Endomycorrhizae further classified in to five types. 1. AM fungi (Arbuscular mycorrhizae) 2. Orchidoid mycorrhizae 3. Monotropoid mycorrhizae 4. Ericoid Mycorrhizae 5. Arbutoid mycorrhizae

9. Acaulospora dan Entrophosphora Glomus, Gigaspora, Scutellospora Spore formation and spore-based taxonomy

10. Vesicular-Arbuscular Mycorrhiza (VAM) Also called as Arbuscular mycorrhiza as vesicles are not formed in all cases. Develop arbuscules and vesicles within root cortical cells . Vesicles Arbuscles Intercellular hyphae form large swellings (vesicles) at ends of hyphae or intercalary Typically rich in lipids & used as storage organs Surrounded by plant cell membrane Typically disintegrate after 2 weeks in plant cell and release nutrients sites where carbohydrates and nutrients are exchanged between cell and VAM hyphae.

11. AM fungi ( Arbuscule mycorrhizae) : Fungi formed AM association with plants may belongs to ascomycetes , basidiomycetes and zygomycetes . All AM fungi are obligate biotrophic , as they are completely dependent on plants for their survival. Many microorganism form symbioses with plants that range from parasitic to mutualistic. Among this the most widespread mutualistic symbiosis is the arbuscular mycorrhizal association. Arbuscular mycorrhizal (AM) symbiosis occurs between the fungi of the Glomeromycota ( Schubler et al., 2001) and majority of terrestrial plants. The phycobiont correspond to 80% of plant species and this association involves an intimate relationship between plant roots and fungal hyphae. This mutualism is manifested in bidirectional nutrient exchange: the fungus is nourished by plant photosynthates and plant mineral nutrition particularly phosphate is enhanced by the fungus (Smith and Read, 1997). AM fungi are obligate biotrophs , depending on living root tissue for carbohydrate supply to complete their asexual life cycle.

12. Spores: These are the "fruiting bodies" of the fungi and are formed both inside the roots and externally in the soil. Spores of diameters ranging from 50 to 400 μ m Depending upon the season and conditions, spores can make up a significant amount of mycorrhizal biomass.

13. Mycorrhiza as root extension The fungus also produces hyphae outside the roots that serve as root extension; thereby increasing the plant’s potential to absorb water and nutrients from the soil.

14. Need to use mycorrhiza Non-native transplants grown in soil and climactic conditions different from the areas where they are planted Areas under heavy environmental stresses

15. Advantages Protects plants from soil-borne diseases by producing healthier seedlings with higher percentage of survival and resistance to plant diseases. Serves as soil conditioner by making the soil porous and alive. Reduces fertilizer application dose. Improves crop and yield. Environment friendly.

16. Different functions played by the arbuscular mycorrhizal (AM) fungi in the physiology and ecology of their host plants.   Mycorrhizal hyphae interconnect roots with soil particles (3), provide direct connections of root systems of different plant individuals (2), and interact with a number of soil microbes (4).

17. Mechanism of Improved plant growth due to VA Mycorrhiza Increased nutrient uptake Synergistic beneficial interaction with other soil micro-organisms Resistance to plant pathogens Better drought tolerance Production of growth promoting substances Stimulate the growth of beneficial microorganisms Improve soil structure – hyphal polysaccharides bind and aggregate soil particles

18. Farm -level VAM root inoculants production A. Materials Required: Sterilized soil Clay / plastic pots Corn / sorghum / raagi / Rice seeds VAM starter inoculants

19. Culturing of AMF The obligate symbiotic nature of the fungus makes axenic cultivation an important challenge for both scientific and practical point of view. Inability to culture AM in the laboratory is the major limiting factor in their application in agriculture. Though AMF has very broad specificity towards plants including various agricultural horticultural and forestry plant species, but the ability to produce AM in bulk quantities is a major bottleneck. AM biofertilizer is currently recommended only for transplanted and nursery raised crops because of the difficulty in inoculum production as well as the bulk requirement of the inoculum. Various methods were developed for mass production of AM fungi world wide.

20. Soil sample + sterile water Hot water Filter and sieve ( 719μm → 250μm → 50μm → 45μm ) Spores separated from soil particles Mix with carrier material Use when required as biofertilizer i ) Isolation A ) Sieving method :

21. B ) Floatation method : Soil sample + sterile water Separate the soil particles using membrane filter Centrifuge ( Density gradient centrifuge = at 3000rpm for 30 min ) Spores separated from soil particles Mix with carrier material Use when required as biofertilizer

22. Mass Production : Being obligate symbionts AM fungi could be mass produced only in the presence of living roots. Since AM fungal associations are universal and have been reported in almost all terrestrial plants, these can be reproduced on a wide range of host plants. There are several techniques reported for mass production of AM inoculum .

23. In-Vivo Culture AM fungi are grown on roots of green house plants and chopped mycorrhizal roots, often mixed with growth media containing hyphae and spores, are used as source of inoculum. Soil could be replaced by inert substances such as vermiculite, perlite, sand or a mixture of these for crude inoculum production.

24. Mass production of VAM

25. 1. Tank for mass multiplication of AM 2. Sprinkling of water in tank with vermiculite 3. Making of furrows to sow maize seeds Method of production

26. Method of production 4. Sowing the seeds in furrows 5. View of the maize sown AM pit 6. Vermiculite contained raised AM infected maize plants •

27. In-Vitro Culture/Axenic Culture Techniques i) Solution culture ii) Aeroponic culture iii) Root organ culture

28. i)Solution culture Involves growing infected roots in aqueous medium enriched with mineral nutrients required for the growth of the roots under controlled biotic and abiotic conditions.

29. ii)Aeroponic culture Involves applying a fine mist of nutrient solutions to colonized roots for AM fungal inoculum production.

30. iii)Root organ culture Use of a modified agar medium (MS rooting medium)/ liquid medium for creation of increased amount of roots from callus tissue and these roots are infected by AM spores or by surface sterilized root bits obtained from mycorrhizal plant.

31. On Farm Mass Production of Vessicular Arbuscular Mycorhiza Method Sterilize soil by heating for 2-4 hours using a big pan or talyasi or by drying under intense heat of the sun for 2-3 days.   Place the sterilized soil in thoroughly cleaned and dry clay pots.   After cooling the soil, place a pinch of root starter inoculants then cover with a thin layer of soil.   Sow 3-5 seeds in each pot.   Grow the plants for three months under normal conditions. Protect the plants from pest and diseases. Stop watering the plants after 3 months. Cut the plants or stalks when they are completely dried. Allow the soil in the pot to dry further.   Remove soil adhering to the roots. Cut the roots finely and save some rot inoculants for future use. Mix the finely cut roots with the soil/ vermi compost from the pot to produce VAM soil inoculants.   Store the root and soil inoculants in sealed plastic bags in a dry and cold place.

32. VAM application Mycorrhizal application goal is to create physical contact between the mycorrhizal inoculant and the plant root. Mycorrhizal inoculant can be sprinkled onto roots during transplanting, worked into seed beds, blended into potting soil, “watered in” via existing irrigation systems, applied as a root dip gel or probed into the root zone of existing plants. Mycorrhizae inoculant can be applied by broadcast thinly on prepared seedbed. Mix into the soil or in case of nursery, cover VAM soil inoculant with a thin layer of soil. The type of application depends upon the conditions and needs of the user. Sow seeds on the seedbed and cover seeds with a thin layer of soil. If necessary, apply fertilizer 15 days after sowing. Do not use chemical fertilizer / fungicide on seedbed before sowing if VAM is to be applied.

33. Application of AM fungi Nursery application 100 g bulk inoculum is sufficient for one m2. The inoculum should be applied a 2-3 cm below the soil at the time of sowing. The seeds/cuttings should be sown/planted above the AM inoculum to cause infection. For polythene bag raised crops 5 to 10 g bulk inoculum is sufficient for each packet. Mix 10 kg of inoculum with 1000 kg of sand potting mixture and pack the potting mixture in polythene bag before sowing. For out-planting 20 g of AM inoculum is required per seedling. Apply inoculum at the time of planting. For existing trees 200 g AM inoculum is required for inoculating one tree. Apply inoculum near the root surface at the time of fertilizer application.

34. Micorrhiza Application Rates Row Crop Application : side dress seed furrows or transplants @ 6 to 9 kg/ha. Incorporate at 2-6 inches (5 to 15cm) below soil surface. Seed Drilling: incorporate into the soil at a depth below the seed. Broadcast and Till : Evenly distribute across seedbed after seeding. Cover the exposed seed and inoculum by harrowing , chain dragging or applying an organic topdressing . Nursery Medium: Evenly blend @ 3 kg/ m 3 . Do not leave inoculum exposed to sunlight.

35. Application : Increase nutrient uptake of plant from soil. P Nutrition and other elements: N, K, Ca, Mg, Zn, Cu, S, B, Mo, Fe, Mn , Cl Increase diversity of plant. Produce uniform seedling. Significant role in nutrient recycling. More tolerant to adverse soil chemical constraints which limit crop production. Increase plant resistance to diseases and drought. Stimulate the growth of beneficial microorganisms. Improve soil structure. Stable soil aggregate – hyphal polysaccharides bind and aggregate soil particles. Increases absorption of phosphate by crops. uptake of zinc also increases. Increases uptake of water from soil. Increases uptake of sulphur from the soil Increases the concentration of cytokinins and chloroplast in plants. They protect plants during stress condition.

36. Benefits : Produce more vigorous and healthy plants. Increase plant establishment and survival at seedling or transplanting. Enhance flowering and fruiting. Increase yields and crop quality. Improve drought tolerance, allowing watering reduction. Optimize fertilizers use, especially Phosphorus. Increase tolerance to soil salinity. Reduce disease occurrence. Contribute to maintain soil quality and nutrient cycling. Contribute to control soil erosion.

37. No mycorrhizal treatment Mycorrhizal treatment No mycorrhizal treatment Mycorrhizal treatment

38. Few mycorrhizal products available commercially

39. Conclusion : Mycorrhizal association is very essential for the plants because it has several benefits like absorption of nutrients, increases drought resistance, enhance plant efficiency in absorbing water and nutrients from soil. Especially, AM fungi are very useful in the agriculture because it serves as biofertilizers as it helps in the absorption of phosphorus, and other nutrient uptake .

40. References : N S subba Rao, Soil microorganisms and plant growth, 3rd edition. Oxford & IBH publishing co. Pvt. Ltd, New Delhi, page no 287-296. Tanuja Sing, S S Purohit and Pradeep Pairhar , Soil microbiology. Agrobios (India), page no 383-405. Himadri Panda and Dharamvir Hota , Biofertilizers and organic farming, 2007 gene- Tech books New Delhi, page no 147-187. Rangaswamy G and Bhagyaraj . Agricultural microbiology and 2nd edition. Prentice- hall of Indian private limited. Dr. H A Modi, Biofertilizers & organic forming, Aaviahkar publishers, Distrubutros jaipur 302003(Raj) India.