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AMAM-FUNGI FROM RHIZOSHPERIC SOIL AND ITS IMPACT ON PRODUCTIVITY

DR. M.L. GUPTA SCIENTIST MICROBIOLOGY AND PLANT PATHOLOGY DIVISION CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS (CIMAP), LUCKNOWLUCKNOW-226015

Mycorrhiza refers to a mutualistic, symbiotic relationship formed between fungi and living roots of higher plants


There are three major groups of mycorrhizae: Ectomycorrhizae and endomycorrhizae. The ectomycorrhizae are generally, association of higher woody perennials. The fungi from intercellular ramifications of mycelium within the host cortex (the Hartig net) and dense hyphal encapsulations (the mantle)

Endomycorrhizae are characterized by fungal penetration of the host cells.  Three types of endomycorrhizae are recognised viz., orchidaceous, ericoid and vesicularvesicular-arbuscular.  The associated endophytes infirst two belong to higher fungi with septate hyphae.


VesicularVesicular-arbuscular mycorrhizae (VAM) or Phycomycetous mycorrhizae are formed by fungi belonging to the order Glomales in Zygomycotina.  The name vesicular-arbuscular (VA) refers vesicularto the formation of typical morphological structures called vesicles and arbuscules in the cortical region of the roots  It is believed that there is exchange of carbon and phosphorus nutrition between the two symbionts for mutual benefits. Six genera viz., Glomus, Acaulospora, Glomus, Acaulospora, Gigaspora, Gigaspora, Scutellospora, Sclerocystis and Entrophosphora have been shown to form mycorrhizal association


VAM are remarkably widespread, distributed geographically throughout the plant kingdom. They occur over a broad ecological range from arctic to desert environments.  Their benefits to the crop plants are well documented. Higher plant growth and yield responses have been reported in many crops such as barley, lucerne, soyabean, corn and mungbean.  VA-mycorrhizae double the efficiency of P VAfertilizers and help the utilization of traditional sources of P fertilizers like bonemeal and rock phosphate more efficiently.


They increase water efficiency of crop, alleviate micronutrient deficiency, alleviate heavy metal toxicity, increase salt tolerance of the crop and reduce diseases of the plants. The primary benefit is however, the more efficient supply of P to the Plants

Palmarosa seedling (i) inoculated with VAM isolate (ii)uninoculated control. Seedlings are three months old. Note the better growth of inoculated seedling

VAM TECHNOLOGY
Interest in mycorrhizal fungi has reached its peak in recent years. The ability of these fungi to produce a dramatic response in plant growth is well documented.  Phosphorus plays a vital role in plant metabolism and nitrogen fixation.  Legumes require high P levels for their growth and effective nodulation. Legume is a cheapest way to enrich tropical soil with nitrogen. Because of the beneficial effects of P on all the nitrogen fixing organism.


P

has a tremendous potential as an indirect source of nitrogen.  VA-mycorrhizae not only help to VAconserve and use phosphorus efficiently but also help in nitrogen nutrition of plants.

VAM fungi has potential importance in the recovery of disturbed lands and these can be effectively used in land reclamation.  They have been conclusively shown to improve revegetation of coal spoil, strip mines, waste areas, road sides and other disturbed habitats. In stress sites, population of VAM fungi provides a nutritional advantage to associated plants in addition to providing resistance to low pH, heavy metal toxicants and high temperatures.  The use of mycorrhizal plants for revegetation at such stressed or disturbed sites has now become a normal practice in many countries.


The use of VAM fungi in afforestation is also gaining imporance. The beneficial effects of inoculating forest nurseries with endomycorrhizae to improve plant growth are well known. Many of the forest trees have been shown to form VA-mycorrhizae. VAAttempts are now being made to employ them as inoculants in forest nurseries to avail their benefits.  Utilization of mycorrhizal technology in commercial production of food, fiber or fuel has been minimal. One of the main reasons is the difficulties in inoculum production.


IDENTIFICATION OF VAM FUNGI


VesicularVesicular-arbuscular mycorrhizal (VAM) fungi are difficult to identify in the absence of knowledge on their complete life cycle. These fungi have also not been cultured on artificial media.  The identification of VAM fungi is therefore done on the basis of morphology of azygospores/chlamydospores/sporocarps and hyphae attached to these structures.


Make the following observations with a compound microscope Spore colour: Spore diameter : Range m; mean m m

Sporocarp (present or absent) if present, diam. Spore contents: (globular, reticulate or granular) Colour of the content Spore wall number Width of each wall ; wall diameter: m

Wall ornamentation present or absent Hypha present or absent. If present Pore diam. Pore occluded? Yes Septum at pore? Yes Type of hyphal attachment, (straight, recurved, funnel shaped, branched, constricted or swollen). Identify the species with the help of descriptions given in the manual. m No No

Glomus

Chlamydospores

Gigaspora

SCUTELLOSPORA

Sclerocystis

ISOLATION OF VAM SPORES FROM THE SOIL


Take 100g of soil Put it in 500ml water and stir well Allow heavier particles to settle down

Decant the suspension through 710

m sieve

Pass the suspension through a series of sieves Viz 425, 250, 106, 75 and 45 m

Collect the residues from 250 and 106 m sieves for sporocarps and largespores and for small spores from 75 and 45 m sieves

Mount them in lactophenol on clean slides and examine them under microscope

Write down the characters for identification of spores

ASSESSMENT OF VAM INFECTION IN ROOTS Collect the roots and wash them well in water

Cut them into 1 cm bits

Put in 10%KOH solution


0

Incubate at 90 C for 2 or 3 hours

Wash the root bits in water (decolourize the coloured roots with H2O2) Acidify them with 5N HCl

Stain the roots in 0.5% trypan blue or cotton blue in lactophenol Mount the root bits in glycerol or lactophenol on clean microscopic slides

Observe for Vesicular/Arbuscular/hyphal infection under microscope.

Studies on mycorrhizal association in Palmarosa


Palmarosa (Cymbopogon martini var. motia) the source of palmarosa oil, was found to be associated with a vesicular-arbuscular mycorrhizal (VAM) fungus. The symbiont Glomus sp. was isolated from the rhizosphere soil, established and multiplied with its natural host palmarosa for the first time. The endophyte appeared to be an undescribed Glomus sp.
Table : Effect of Glomus sp. on Growth response and mineral uptake in Palmarosa
S. No. Treatment No. of Tillers 1. 2. C.D. % Glomu sp. Control 5% 1% 0.1% 22 6 5.996 9.994 18.597 Growth response Height (cm) 275 150 38.430 63.730 119.190 Dry wt (g) 130 20 21.440 35.560 66.500 Soil P (ppm) 6 10 2.531 4.198 7.852 K (ppm) 24 36 0.793 1.316 2.462 Mineral uptake Plant P (%) 0.18 0.11 0.028 0.046 0.086 K (%) 1.75 1.40 0.302 0.502 0.938

Thus experimentals showed that the mycorrhizal associations enhanced growth, total biomass and mineral uptake especially phosphorus and potassium significantly over control

Palmrosa root showing extensive colonization of endophytic mycorrhiza Root cortex showing cluster of spores of Glomus sp. Spore of Glomus sp. With oil globules Intramatrical vesicles of Glomus sp. in the root cortex of infected root Arbuscules of Glomus sp. in cortex of infected roots (scale bar = 20 m)

Another VAM-fungus Glomus aggergatum have been isolated from rhizosphere soil of palmarosa growing at CIMAP forms. This symbiotic fungus is successfully established, maintained and multiplied with its natural host for further studies. Details morphological characters of the symbiont have been studied and recorded as a new report on palmarosa. The glasshouse experiments showed that inoculation of palmarosa with Glomus aggregatum caused a two fold growth and three fold biomass production as compared to non mycorrhizal control plants.
Table : Effect of Glomus aggregatum on the growth, biomass production and phosphrus uptake of palmarosa
Height (cm) Treatments Glomus aggregatum Control S.E. +/155 No. of Tillers 18.80 Stem thickness (mm) 19.00 Fresh wt (g) Phosphorus content Plant (%) 220 0.1650 Soil (ppm) 5.60

80 8.324

7.80 1.257

8.00 0.894

70 11.726

0.1080 0.0041

9.20 0.129

Effect of Glomus aggregatum, G. fasciculatum and G. mosseae were evaluated on the productivity of Palmarosa var. Trishna, Lemongrass var. Praman, and Citronella var. Manjusha in the field condition along with proper control
Table: Effect of VAM-fungi on the productivity and essential oil yield of aromatic grasses
Treatment S. No. Biomas s Kg/plot GA GF GM Control 5% 1% 7.20** 6.50* 5.80 4.80 1.23 2.09 Palmarosa % oil yield 0.62** 0.62 * 0.60 0.60 Total oil content 44.64** 40.30 * 34.80 28.80 6.17 9.23 Lemongrass Biomass Kg/plot 11.00** 9.20 * 7.80 6.60 2.36 4.19 % oil yield 0.64 0.64 0.62 0.64 Total oil content 70.40** 58.88 * 48.36 42.24 16.64 27.23 Biomass Kg/plot 5.50** 4.60 * 3.60 2.90 1.06 2.43 Citronella % oil yield 0.95 0.95 0.94 0.92 Total oil content 52.25** 43.70 * 33.84 26.68 8.45 17.12

1. 2. 3. 4. C.D .

Results indicated that all the VAM fungus are capable of to enhance the total biomass yield of the plants. However, among them GA responded well and enhanced the herb and oil yield significantly over the control. Besides, it is surprising that GA has enhanced the herb as well as oil yield of citronella to more than 80%. reason was noted that control plants were found suffering with serious yellowing disease which was drastically reduced the total herb yield in control treatment.

Table: Effect of different VAM-fungi on the growth and productivity of peppermint (Mentha piperita L.) Height Treatments (cm) GA GF GM C C.D. 5% 1% 42.20 47.10 39.30 35.40 2.14 3.32 Shoot fresh wt (g) Shoot dry wt (g) % oil Content 0.62 0.61 0.60 0.60 0.09 0.13 Root colonizatio n (%) 49.00 67.70 58.40 4.77 6.94 Chlamydospore s population (100g-1 soil) 664 408 684 41.90 61.00

68.40 25.30 (131.1) 72.60 27.40 (145.3) 55.60 19.90 (87.80) 29.60 2.84 3.99 10.60 3.04 2.26

Where as C= Non mycorrhizal control GA= Glomus aggregatum, GF= G. fasciculatum, GM= G. Mosseae (..) Value in the parenthesis indicate the percent increase over control

Table: Effect of different VAM-fungi on the nutrient uptake of peppermint


Nutrient uptake (ppm) Treatment GA GF GM C C.D. 5% 1% P 2.80 (100.00) 3.20 (128.60) 2.60 (85.70) 1.40 0.35 0.48 K 16.10 (73.10) 20.50 (120.40) 16.30 (75.30) 9.33 0.88 1.23 Zn 0.76 (46.20) 0.95 (82.70) 0.68 (30.70) 0.52 0.08 0.11

Note:- Values in the parenthesis indicate the percent increase over control

Table: Influence of G. fasciculatum on the growth, total herb yield and root colonization of menthol mint cultivars under field conditions
Mint cultivars Height (cm) Treated Control Shivalik Treated Control Gomati Treated Control C.D. at P= 0.05 P= 0.01 M V M V 41.25 36.30 63.35 53.80 65.80 57.20 2.24 2.74 3.10 3.80 Fresh herb Dry herb yield yield (q/ha) (q/ha) 83.855 (26.53) 66.273 90.617 (52.27) 59.510 100.085 (29.82) 77.093 8.548 10.485 11.794 NS 26.238 20.363 26.714 16.976 29.117 22.852 0.24 0.29 0.33 NS Essential oil yield (%) 0.613 0.607 0.640 0.620 0.593 0.563 0.010 0.012 0.014 0.017 VAM colonization (%) 57.20 13.20 68.20 19.60 60.60 16.80 2.24 2.76 3.09 3.82

Hyb.77

Where as :- M = Mycorrhiza Glomus fasciculatum, V = Variety/cultivar NS = Non-significant () = Values in the parenthesis indicate the percent increase over the control - treatment

Table: Effect of different G. fasciculatum on the nutrient uptake of menthol mint cultivars
Nutrient status in soil (mg/kg) Mint cultivars Hyb.77 Treated Control Shivalik Treated Control Gomati Treated Control C.D. at P= 0.05 P= 0.01 M V M V N 48.50 57.40 50.90 54.10 48.50 50.40 NS NS NS NS P 20.90 (-36.66) 33.00 21.60 (-24.21) 28.50 17.80 (-27.35) 24.50 4.15 NS NS NS K 66.50 70.50 64.50 77.50 64.50 66.00 NS NS NS NS Plant uptake (kg/ha) N 31.345 (32.34) 23.685 33.401 (75.78) 19.001 33.477 34.559 4.997 6.113 6.881 NS P 4.772 (15.77) 4.122 4.934 (66.46) 2.964 6.211 (71.34) 3.625 1.082 NS 1.504 NS K 35.110 (13.14) 31.032 36.864 (39.68) 21.726 36.928 (22.02) 30.263 4.425 NS 6.146 NS

() Values in parenthesis indicate the percent increase or decrease (-) over the control treatment

Table: Effect of dual inoculation of VAM-fungi and growth promoting bacterium on the growth and nutrient uptake of palmarosa
Treatment Plant height (cm) Fresh biomass yield (g/plant) Shoot Control GA PF GA+PF 64 81 80 94 33 46 61 67 Root 14 23 29 35 Phosphatase activity ( g/100mg/30mt) Shoot 197 222 229 307 Root 83 127 110 132 Phosphorus content (mg/g dry wt) Shoot 0.86 1.01 0.92 1.09 Root 0.88 1.27 1.20 1.50 75 86 406 676 % root colonization VAM population (per-100g soil)

GA = Glomus aggregatum; PF = Pseudomonas fluorescent Experimental findings indicated that dual inoculation promoted 14% higher colonization of palmarosa roots than GA alone. It also increased the phosphates activity (59%) and phosphorus content (71%) in the roots of host plant. Similarly PF incorporation along with GA enhanced the herb yield by 45% more than sole application of GA. Experimental findings, thus, concludes that dual application of micro-organisms seems to be more effective to improve the productivity of palmarosa than their individual applications.

Interaction effect of some useful micro organism on the root colonization, herb yield and essential oil content of Indian basil (Ocimum basilicum)
Treatments Root colonization (%) 71.2 89.4 (23.8) 56.0 Spore population (100-1 soil) 680 860 600 Shoot fresh wt per pot (g) Essential oil contents (%)

Control BM AA GA GA+BM GA+AA

96.2 134.2 (39) 104.7 (9) 129.7 (35) 144.5 (50) 112.0 (16)

0.40 0.45 0.50 0.45 0.45 0.45

BM = Bacillus megatherium, AA = Aspergillus avamori, GA = Glomus aggregatum (--) indicate percent higher yield in comparison to control

Mycorrhizal association of Indian basil. A. Healthy plant, B. Root colonization of basil showing vesicles and spores of Glomus aggregatum

Effect of VAM-fungi on the growth and productivity of Catharanthus roseus


A glass house experiment was conducted to evaluate the effect of various VAM-fungi viz. Glomus aggregatum (GA); G. fasciculatum (GF); G. mosseae (GM); G. intraradices (GI) and G. versiforme (GV) on the growth and biomass production of this plant.
Growth parameters Treatments Fresh biomass (g) Shoot Control GA GF GM GI GV 38.7 52.3 51.0 61.0 56.0 61.6 Root 26.3 38.6 35.0 55.6 40.0 44.0 Dry matter (g) Shoot 13.3 16.0 15.7 18.7 16.0 18.0 Root 8.8 12.0 10.3 17.8 13.0 14.3 61 48 69 67 65 Root colonization (%)

The finding indocated that all the VAm-fungi are capable of enhancing the growth and total biomass yield of this plant. However, maximum herb yield was found in G. mosseae (36.6%) followed by GV (36%), GA (35.1%), GI (30%)and GF (24.1%) over control. A similar trend was also observed in % root colonization of the plant. Thus, higher root colonization by GM (69%) may be a reflection of increased biomass yield of C. roseus.

A portion root showing colonization by VAM -fungi

Table: Interaction of VAM-fungi and root-knot nematode on total biomass yield of black henbane (Hyoscmus niger L.)
Treatment VAMroot infection (%) 75 45 53 12.05 17.13 VAM-spore population (100g-soil) 370 210 230 39.05 55.51 Dry wt. of plant (g) Root Shoot Total

Control Nematode (Mi) GA+Mi GF+Mi GM+Mi C.D 5% . 1%

10.70 6.26 10.70** 7.90** 7.33** 0.76 1.08

49.36 27.80 36.80** 33.80** 33.00** 2.30 3.27

60.06 34.09 47.50 41.70 40.33 -

GA = Glomus aggregatum GF = Glomus fasciculatum GM = Glomus mosseae Mi = Nematode-Meloidogyne incognita

Table: Effect of different VAM-fungi on the nematode (M. incognita) reproduction on black henbane Treatments Nematode population in total root 14066 10755** 12889 12427* 5% 1% 1382.36 1965.06 Nematode population in total soil 21400 11600** 15200 18760* 1736.60 2468.75 Total nematode population (root + soil) 35466 22355 28089 31187 Reproduction factor (RF) RF = PF/PI 7.09 4.47 5.62 6.24 -

Control Nematode (Mi) GA+Mi GF+Mi GM+Mi C.D.

PI = Initial nematode population PF = Final nematode population

Antagonistic impact of vesicular-arbuscular mycorrhizal fungi on Meloidogyne incognita population development in Japanese mint
Treatment Control Mi GA GA + Mi GF GF + Mi GM GM + Mi GA+GF+GM GA+GF+GM+Mi C.D. 5% 1% % root colonization 45.5 40.3 60.0 55.2 65.5 62.0 80.0 72.5 12.05 17.13 Fresh biomass yield (g) Root 192 147 194 172 196 180 198 185 200 188 2.14 1.72 Shoot 203 138 204 150 206 153 210 203 218 172 2.81 1.54 Total 395 285** (-27.8) 398** (+0.7) 322** (-18.5) 402** (+1.7) 333** (-15.7) 408** (+3.2) 388** (-10.8) 418** (+5.8) 360** (-8.9) Nematode population Root 113190 79840 54352 46080 48800 1020.43 1302.72 Shoot 15600 8000 7200 5600 6000 408.27 631.18 Total 128790* (25.75) 87480* (17.49) 61552* (12.31) 51680* (10.33) 54800* (10.96) % oil content 0.51 0.38 (-25.5) 0.52 (+1.9) 0.40 (-21.5) 0.53 (+3.9) 0.41 (-19.8) 0.54 (+5.8) 0.48 (-5.9) 0.54 (+5.9) 0.46 (-9.8) 0.01 0.22

GA = Glomus aggregatum; GF = Glomus fasciculatum; GM = Glomus mosseae Mi = Meloidogyne incognita ()** Parenthesis indicate-percent increase (+) or decrease (-) over control.; ()* RF value

Damping-off of henbane seedlings and its management


Interaction of Glomus aggregatum (GA), Bacillus megatherium (BM), or its combination with Pythium aphanidermatum (PA) on the growth and total biomass yield of Egyptian henbane
Treatments Plant height (cm) Total fresh biomass (g) Shoot 1. Control 2. GA 3. BM 4. GA+BM 5. PA 70 78 72 80 62 73 66 76 165 (42.24) 208 (26.0)* (79.3)** 178 (7.9)* (53.4)** 236 (43.0)* (103.4)** 116 180 (9.1)* (55.2)** 160 (-3.0)* (37.9)** 214 (29.7)* (85.5)** Root 28 36 30 40 18 30 26 32 % root colonization 62 78 42 60 Spores population (100-1soil) 660 900 480 580

6. GA+PA 7. BM+PA 8. GA+BM+PA

()values in the parenthesis are the percent increase(+) or decrease (-) in shoot weight when compared with treatment 1(*) or 5 (**)

Interaction of beneficial micro organisms with Fusarium solani and its effect on the growth, herb and essential oil yield of Mentha arvensis
Treatments Height (cm) Fresh wt (g/pot) Oil contents (%) 0.78 0.80 0.34 0.82 0.82 0.80 0.80 0.81 0.81 Total oil yield (ml/pot) 0.92 0.62 0.14 1.54 1.28 1.22 1.07 0.97 1.13

CI CII FS GM PF TR GM+FS PF+FS TR+FS

48 46 42 56 52 52 50 48 51

118 78 40 188 156 152 134 (40.29) 120 (30.0) 140 (8.57)

FS = Fusarium solani, GM = Glomus mosseae, PF = Pseudomonas fluorescens, TR = Trichoderma viride (..) percent loss over their alone treatment CI = Control with unsterilized soil, CII = Control with sterilized soil

Conc. (%) Treatment Benomyl 0.05 0.10 Karathane 0.05 0.10 Captan 0.05 0.10 Vitavax 0.05 0.10 Thiram 0.05 0.10 Dithane M-45 0.05 0.10 Brassicol 0.05 0.10 Nuan 0.05 0.10 Eucalox 0.05 0.10 Roger 0.05 0.10 Control C.D. 5% 1%

Tillers plant-1 12.5 7.5 11.0 10.0 10.5 11.0 17.5 10.0 8.5 10.5 8.0 8.5 12.0 10.5 7.5 7.0 15.0 11.0 7.0 7.0 10.5 1.206 1.254

Height (cm) 185.0 132.0 124.3 86.1 157.7 120.5 224.4 102.5 107.5 136.0 132.0 129.0 147.3 136.7 127.0 125.5 154.8 136.5 122.7 119.3 133.25 12.120 12.608

Fresh wt (g) 91.50 65.00 46.50 45.20 92.50 55.00 94.50 57.00 42.50 76.00 47.50 46.90 88.50 47.20 41.00 38.50 99.50 78.00 52.00 49.00 74.25 8.703 9.049

Spores (100g soil-1) 510.00 230.00 250.00 182.00 450.00 295.00 570.00 248.00 320.00 480.00 150.00 162.00 460.00 275.00 220.00 190.00 585.00 390.00 200.00 190.00 367.50 55.038 57.224

Root colonization (%) 82.50 54.20 62.40 47.60 81.00 65.60 88.50 55.00 68.60 81.00 38.80 39.20 80.50 60.50 51.50 48.00 90.00 78.00 50.20 50.00 74.25 7.015 7.294