ARBUSCULAR MYCORRHIZAL STATUS OF CARICA PAPAYA L. AS INLFUENCED BY ITS PHENOLOGY

Transcription

1 CHAPTER - 4 ARBUSCULAR MYCORRHIZAL STATUS OF CARICA PAPAYA L. AS INLFUENCED BY ITS PHENOLOGY

2 UNTRODUCTION: Phenology may influence the temporal pattern of nutrient demand (Ronson, 1987). Phosphorous demand, defined as the rate of P uptake from the soil needed to give optimum response, may thus be higher in the modem cultivars during grain filling stage because they must accumulate a given amount of phosphorous into reproductive tissues in a relatively short period of time. In contrast, a hydroponic experiment of Lolium peronne exhibited that the optimum nutrient solution concentration was actually less for later stages of growth than for the seedling stages (Breeze et al., 1985). Although fruit and seed production may be more expensive processes than vegetative growth a far as mineral are concerned, root system development at the time of reproduction may be proportionally greater in relation to demand than during early seedling growth. In addition, the extent to which the nutrient expense of reproduction must be borne by root uptake depends on how much can be reallocated from existing stores within the vegetative portions of the plant. Chapin (1980) reported that approximately 50% of leaf phosphorous may be reallocated prior to senescence. Arbuscular mycorrhizal fungi can enhance plant uptake of inorganic phosphorus from soil through hyphal scavenging of soil volumes that are not accessed by roots (Joner et al., 2000). The fungi exploits the same pools of inorganic phosphorus that are exploited by roots (Bolan,1991), but bypass the slow process of diffusion by translocating soluble inorganic phosphorus from non rhizosphere soil to their host through extraradical hyphae (Sanders and Tinker, 1971). However, when the plants grow under P.-deficient conditions, plant roots secrete organic acids and acid phosphatase (Tadano et al., 1993). Mycorrhizal colonization could also alter inherent phosphorous supply by either 149

3 increasing the activity of alkaline phosphatase in roots and in the rhizosphere (Allen et al., 1981b, Dodd et al., 1987) or by reducing root acid phosphatase activity (Azcon et al., 1982). However the ability of the crop plants to secrete phosphatases has not been compared under same growth conditions neither its arbuscular mycorrhizal status has been assessed in relation to phonology. Therefore, in the present chapter, comparative studies on four dioecious varieties of Carica papaya L. was undertaken for one growing season, to ascertain information on arbuscular mycorrhizal association and root phosphatase activity in relation to phenology of the host plant. MATERIALS AND METHODS: Study site and management regimes- Papaya plantations in agricultural farm located at Old Goa (North Goa) was selected for the study. The soil at this site was well drained, dark grey brown to dark brown gravelly sandy loam to sandy clay loam with medium water holding capacity. The study was carried from June 2001 to September The maximum and minimum temperatures recorded during this period were 31 C and 25.5 C respectively with relative humidity ranging from 82-87%. Rainfall was ranging from 120mm (September) mm (July). Four dioecious varieties viz., CO-1, CO-2, Honey Dew and Washington were planted in monoculture. Papaya plantations were managed conventionally. Inorganic fertilizer (19:19:19) was applied in six split doses per year at an interval of two months. Papaya plants received 250g N, 250g P205 and 50g K20 per plant per year. So during the study period, inorganic fertilizer was applied once e. in September. Plantations were irrigated twice a week all the year round except in monsoons. However, the entire study was carried 150

4 out in the wet season and therefore no irrigation was carried out during this period. The weed species mostly comprised of grasses that were present at the periphery. However the weed species did not interfere with sample collection procedures as the mulching at the base of the plant was first cleared and then the samples were collected. Also it was observed that Carica papaya L. produces numerous feeder roots on the surface in response to soil moisture. So the collections were carried out at the depth of 0-25 cm through out the study period. Collection of samples- Generally papaya seedlings are raised in the nursery for two months and than they are transplanted in the field. The early vegetative stage mainly comprises of tap root system with feeble primary branches making it difficult to collect root (feeder roots) samples to carry out mycorrhizal studies. Hence the study was initiated during late vegetative stage (here after referred to as `vegetative stage') when the plants were six months old plants and exhibited extensive root system with ample of feeder roots available for collection. Five stages of development viz., vegetative (first week June), flowering- I (first week of July), flowering-ii (last week of July), fruiting I (mid August) and fruiting II (mid -September) were considered for the study with the time of sampling given in parenthesis. In Carica papaya L., the male and female plants of could be distinguished only at the flowering stage-i. Therefore from flowering stage-i onwards, male and female plants were sampled separately for each variety. At flowering stage -I, only male plants were flowering whereas, at flowering stage II, both male and female plants were flowering. Fruiting stage -I marked the onset of fruiting in female plants while the male plants at 151

5 this stage were flowering. Fruiting stage-ii marked the late fruit bearing stage of Carica papaya L. and this time the fruits were fully matured while the male plants were still flowering. Sampling procedures were carried out according to Tews and Koske (1986) except that the core size was bigger (15 cm in diameter). This was carried out to avoid non-normal distribution of spores recorded in counts from small core samples (St. John and Koske, 1988). During the vegetative stage, two healthy plants per variety were randomly selected for the collection of rhizosphere soil and root samples. However, from flowering stage onwards, four plants were sampled per variety (2 male and 2 females). For each plant, three random soil cores were collected from within 60 cm of each plant, each at the depth of 0-25cm. Sub-samples collected from both plants were then combined to make composite sample after thorough mixing. Composite samples for male plants and female plants were made separately. From each composite sample, 5 sub-samples were made for quantification of spore density, 2 sub samples for nutrient analysis and two sub samples for establishment of pot cultures. Roots were packed separately for assay of phosphatases and for estimation of root colonization per plant in the field. Root samples collected for assay of phosphatases, were transported in ice to the laboratory. These root samples (feeder roots) were washed in ice cold water and rinsed with double distilled water, packed in polyethylene bags, labeled and stored at -80 C. For estimation of root colonization and establishment of pot cultures, the root samples of both the plants were combined. From this composite sample, five sub-samples were utilized for estimation of degree of root colonization and two sub-samples were utilized for pot cultures. 152

6 Establishment of pot cultures- Baiting of native arbuscular mycorrhizal fungi were carried out by using open pot cultures (Gilmore, 1968). Two pot cultures were maintained per variety during the vegetative stage and thereafter four pot cultures (two for male plants and two for female plants) were maintained per variety. The roots of host species were checked for arbuscular mycorrhizal colonization after 45 days. Pots showing successful mycorrhization were maintained for period of six months and application of water was reduced at final three weeks to maximize spore production (Menge, 1982). At the end of 6 months the plants were cut near the base, the cultures were air-dried and checked for the presence of spores. Spores isolated from pot cultures were used for verifying the identification of arbuscular mycorrhizal fungi recovered from original field samples. Soil sample analysis- Two rhizosphere soil samples per variety (one sample/plant/ variety) were employed for analysis of 9 edaphic factors during the vegetative stage. From the flowering stage I to fruiting stage II, four soil samples per variety at each growing stage were employed for analysis (2 for males and for 2 females). Samples were analysed separately for male and female plants of a particular variety at each growth stage. Soil ph was measured in 1:2 soil water suspension using ph meter (LI 120 Elico, India). Electrical conductivity was measured at room temperature in 1:5 soil suspension using Conductivity meter (CM-180 Elico, India). Standard soil analysis techniques viz., micro-kjeldahl method (Jackson, 1971) and Bray and Kurtz method (1945) were employed for determination of total nitrogen and available phosphorus respectively. Available potassium was estimated by ammonium acetate method (Hanway & Heide!, 153

7 1952) using Flame Photometer (Systronic 3292). Available zinc, copper, manganese and iron were quantified by DTPA-CaCl2- TEA method (Lindsay & Norvell, 1978) using Atomic Absorption Spectrophotometer (AAS 4139). Estimation of root colonization by arbuscular mycorrhizal fungi Roots were cleared in 10% KOH, acidified in 1 N HCL and stained in 0.05% trypan blue in lactoglycerol (Phillips and Hayman, 1970). Total root colonization and length of the root colonized by hyphae, arbuscules and vesicles by arbuscular mycorrhizal fungi were estimated by grid intersection method (Mc Gonigle et al., 1990). Assay of phosphatase enzyme activity - The study comprised of two repetitions / variety / developmental stage and each repetition had five replications. Activities were assayed separately for males and females from flowering stage I onwards. One gram of root tissue was weighed for each repetition. Methods for quantification of activity of phosphatases were standardized for Carica papaya L. based on procedures provided by Kapoor et al. (1988) and Sukhada (1992). Extraction of enzyme - Enzyme was extracted by macerating 1g of detached root tissue at 4 C using 20 ml of phosphate (0.1 M KH2 PO4, ph 6.6) buffer. The homogenate was filtered through muslin cloth and the filtrate was centrifuged at 5000 r. p. m for 15 min using a cooling centrifuge (Remy C-24). The supernatant was stored at 4 C and further employed for protein estimation of enzyme and assay of phosphatases. Protein estimation - Protein content of the enzyme extract was estimated by 154

8 Lowry's method (Lowry et al., 1951) using bovine serum albumin as standard. Assay of phosphatases - For alkaline phosphatase activity, 5lig protein equivalent enzyme extract was incubated with 2 ml of 15 mm p-nitrophenyl phosphate (pnpp) and 0.8 ml 0.25 m Tris HCI (ph 9.8). For acid phosphatase activity, 5lig protein equivalent enzyme extract was incubated with 2 ml of 15 mm p-nitrophenyl phosphate (pnpp) and 0.8 ml 0.25 m Sodium acetate (ph 6.0). The reactions in both the above mentioned cases were terminated by adding 2 ml of 1N NaOH. For each repetition, T'o (zero time of incubation) and T'30 (30 minutes after incubation) were taken separately for alkaline and acid phosphatase activity. Optical density (0.D) was read at 410 rim using uv-1201 Shimadzu spectrophotometer against a blank solvent (distilled water). Phosphatase activity was expressed in terms of n moles of p-nitrophenol released / min/ lig protein. Quantification of spore density of arbuscular mycorrhizal fungi Spores and sporocarps of arbuscular mycorrhizal were isolated by wet sieving and decanting method (Gerdemann and Nicolson, 1963) and quantification of spore density of arbuscular mycorrhizal fungi was carried out using method described by using Gaur and Adholeya (1994). Identification of arbuscular mycorrhizal fungi Diagnostic slides containing intact and crushed spores and sporocarps of arbuscular mycorrhizal fungi were prepared in polyvinyl alcohol lactoglycerol (PVLG) (Koske and Tessier, 1983). Spore morphology and wall characteristics were considered for the identification of arbuscular mycorrhizal fungi and these 155

9 characteristics were ascertained using compound microscope, Leica WILD MP3 and Nikon E 800 Arbuscular mycorrhizal fungi were identified to species level using bibliographies provided by Almeida and Schenck (1990), Morton and Benny (1990), Schenck and Perez, (1990), Wu (1993), Bentivenga & Morton (1995), Walker and Vestberg (1998), Redecker et al., (2000b) and Morton and Redecker (2001). Taxonomic identification of spores was also carried out by matching the descriptions provided by International Collection of Vesicular Arbuscular Mycorrhizal fungi ( invam.caf.wvu.edu ). Names and epithets of arbuscular mycorrhizal fungi were followed according to recommendations of Walker and Trappe (1993). Diversity indices- Species richness per growth stage is the mean number of arbuscular mycorrhizal fungal species recovered at any particular growth stage. In addition species richness was calculated for each variety. It was recorded as number of species of arbuscular mycorrhizal fungi associated with each variety during different stages of development of Carica papaya L. (Beena et al., 2000). Frequency of occurrence- Frequency of occurrence of arbuscular mycorrhizal fungi was calculated using the following formula (Beena et al,2000 ). Frequency - (%) Number of soil samples that possess spores of particular species Total number of soil samples analyzed x

10 Relative abundance- Relative abundance of arbuscular mycorrhizal fungi was calculated using the following formula (Beena et al., 2000). Number of fungal spores of particular species Relative = x 100 abundance (%) Total number of spores of all species Statistical analysis Data on root colonization and spore density of arbuscular mycorrhizal fungi and root phosphatase activity was subjected to one way analysis of variance in order to investigate the variations in these parameters with respect to four varieties of Carica papaya L. during different developmental stages. Prior to analysis of variance, root colonization values were subjected to arcsine transformations. Pearson's correlation test was performed to assess the relationship between root colonization, spore density, root phosphatase activity and edaphic factors. Additionally, multiple linear regression analysis was carried out to determine the relationship between root colonization and root phosphatase activity at each stage of development. Data was statistically analyzed using mstac package. RESULTS: Edaphic factors- Data on edaphic factors recorded during the different growth stages of Carica papaya L. is presented in Table 42 Table 50. The soil ph was acidic, while electrical conductivity was ranging from normal to satisfactory. Available phosphorus recorded low levels in the tizosphere of Carica papaya 157

11 TABLE 42 : Comparative account of the edaphic factors in Carica papaya L. varieties during vegetative stage. VARIETIES * EDAPHIC FACTORS ph E.0 Total N Available P Available K Zn Cu Fe Mn (m mosicm) (%) (Kg/Ha) (Kg/Ha) (ppm) (pppm) (ppm) (ppm) CO (0.218) (0.001) (0.002) (0.861) (5.486) (0.000) (0.088) (0.451) (1.337) CO (0.165) (0.032) (0.001) (0.014) (4.448) (0.001) (0.841) (0.469) (1.580) Honey dew (0.222) (0.035) (0.000) (0.028) (3.231) (0.002) (0.457) (0.587) (1.452) Washington (0.234) (0.033) (0.008) (0.027) (4.520) (0.003) (0.358) (0.521) (1.741) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1 SE.

12 TABLE 44 : Comparative account of the edaphic factors in Carica papaya L. varieties during flowering stage I - Female Plants. VARIETIES * EDAPHIC FACTORS ph EC Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (Kg/Ha) (ppm) (ppm) (ppm) (ppm) CO (0.324) (0.003) (0.000) (0.230) (5.891) (0.000) (0.088) (0.517) (1.456) CO (0.189) (0.004) (0.001) (0.415) (5.691) (0.004) (0.564) (0.621) (1.880) HONEY DEW (0.199) (0.000) (0.001) (0.412) (4.213) (0.002) (0.234) (0.238) (1.691) WASHINGTON (0.432) (0.006) (0.001) (0.514) (2.458) (0.002) (0.224) (0.528) (1.452) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1SE.

13 TABLE 45 : Comparative account of the edaphic factors in Carica papaya L. varieties during flowering stage II - Male Plants. VARIETIES * EDAPHIC FACTORS ph EC Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (Kg/Ha) (ppm) (ppm) (ppm) (ppm) CO (0.223) (0.002) (0.005) (0.254) (5.841) (0.258) (0.854) (0.541) (0.419) CO (0.176) (0.000) (0. 001) (0.221) (3.662) (0.451) (0.236) (0.478) (0.514) HONEY DEW (0.267) (0.001) (0.001) (0.514) (5.691) (0.328) (0.589) (0.657) (0.369) WASHINGTON (0. 342) (0.002) (0.002) (0. 247) (5.999) (0.963) (0.328) (0.238) (0.253) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1 SE.

14 TABLE 46 : Comparative account of the edaphic factors in Carica papaya L. varieties during flowering stage II - Female Plants. VARIETIES * EDAPHIC FACTORS ph EC Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (Kg/Ha) (ppm) (ppm) (ppm) (ppm) CO (0.139) (0.002) (0.001) (2.231) (3.974) (0.284) (0.412) (3.526) (1.364) CO (0.248) (0.008) (0.001) (2.561) (9.231) (0.346) (0.238) (0.512) (2.564) HONEY DEW (0.246) (0.001) (0.002) (3.381) (6.210) (0.542) (0.451) (0.315) (5.230) WASHINGTON (0.547) (0.005) (0.001) (4.691) (2.310) (0.651) (0.221) (0.364) (2.658) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1SE.

15 TABLE 47 : Comparative account of the edaphic factors in Carica papaya L. varieties during fruiting stage I - Male Plants. VARIETIES * EDAPHIC FACTORS ph EC Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (Kg/Ha) (ppm) (ppm) (ppm) (ppm) CO (0.471) (0.000) (0.002) (0.258) (2.440) -(0.521) (0.478) (2.338) (1.857) CO (0.287) (0.005) (0.001) (0.238) (0.651) (0.612) (2.231) (3.010) (3.008) HONEY DEW (0.351) (0.002) (0.000) (0.324) (0.257) (0.478) (0.547) (1.991) (2.847) WASHINGTON (0.338) (0.311) (0.005) (0.561) (0.541) (0.366) (2.569) (0.654) (2.543) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1SE.

16 TABLE 48 : Comparative account of the edaphic factors in Carica papaya L. varieties during fruiting stage I - Female Plants. VARIETIES * EDAPHIC FACTORS ph EC Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (Kg/Ha) (ppm) (ppm) (ppm) (ppm) CO (0.220) (0.000) (0.002) (0.146) (2.254) (0.111) (0.221) (0.222) (2.382) CO (0.213) (0.001) (0.001) (0.591) (1.653) (0.332) (0.114) (0.364) (3.006) HONEY DEW (0.337) (0.003) (0.000) (0.601) (1.189) (0.441) (0.189) (0.395) (1.943) WASHINGTON (0.311) (0.01) (0.003) (0.412) (2.003) (0.255) (0.112) (0.238) (1.965) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1SE.

17 TABLE 49 : Comparative account of the edaphic factors in Carica papaya L. varieties during fruiting stage II - Male Plants. VARIETIES *EDAPHIC FACTORS PH E. C Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (Kg/Ha) (ppm) (ppm) (ppm) (ppm) CO1 (1.456) (0.112) (0.002) (0.001) (0.741) (2.111) (0.011) (0.031) (0.345) CO2 (1.067) (0.118) (0.000) (0.006) (0.881) (1.999) (0.012) (0.054) (0.654) HONEY DEW (1.654) (0.231) (0.003) (0.017) (0.451) (1.235) (0.014) (0.064) (0. 342) WASHINGTON (2.890) (0.328) (0.005) (0.001) (0.324) (1.458) (0.021) (0.028) (0.261) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1SE.

18 TABLE 50 : Comparative account of the edaphic factors in Carica papaya L. varieties during fruiting stage II - Female Plants. VARIETIES *EDAPHIC FACTORS ph E. C Total N Available P Available K Zn Cu Fe Mn (m mhos/cm) (%) (Kg/Ha) (K9410 (PPm) (PPm) (PPm) (PPrn) CO (0. 220) (0.001) (0.002) (0.772) (2.788) (0.041) (0.025) (0.047) (1.368) CO (0.197) (0.005) (0.003) (0. 698) (2.541) (0.021) (0.028) (0.052) (0.225) HONEY DEW (0. 218) (0.001) (0.002) (0.622) (2.232) (0.040) (0.058) (0.327) (1.584) WASHINGTON (0. 221) (0.001) (0.002) (0.715) (2.085) (0.052) (0.048) (0.234) (1.631) *Values presented are mean of two readings; Values in the parenthesis indicates ± 1 SE.

19 L. through out the study except for few instances during fruiting stage-i and fruiting stage-ii (Table 50 and Table 51) whereas, total nitrogen content of the soil was ranging from low (0.28%) to optimum (0.51%) except for few instances, higher values were recorded (Table 49 and Table 50). Potassium levels were mostly ranging from optimum (87 Kg/ Ha) to high (200 Kg/Ha) and only at few sampling stages, low (72-80 Kg /Ha) (Table 43, Table 47, Table 48and Table 49) to very low (58 Kg/ Ha) (Table 43) levels were recorded. Micronutrients in general were present in high concentrations. I> Root colonization of arbuscular mycorrhizal fungi- All the root samples collected during the study period exhibited the presence of arbuscular mycorrhizal colonization which was characterized by the presence of hyphae, hyphal coils, arbuscules and vesicles. Arbuscular mycorrhizal colonization was predominant during the study period (Platel2 a - f). It was observed that the root colonization of arbuscular mycorrhizal fungi varied within the varieties at any particular stage of development of Carica papaya L. Data on total root colonization and root length colonized by hyphae, arbuscules and vesicles of arbuscular mycorrhizal fungi during different stages of development is depicted in Fig. 18, Fig.19, Fig.20, Fig.21 and Fig. 22. a) Vegetative stage: The hyphal (C.D = 3.488; P= 0.05), arbuscular (C.D = 1.202; P= 0.05), vesicular (C.D = 2.599; P= 0.05) and total root colonization (C.D = 4.067; P= 0.05) of arbuscular mycorrhizal fungi varied 158

20 PLATE 12: Arbuscular colonization during the flowering stages of Carica papaya L. intense colonization in Honey dew (a, c, e) and Washington variety (b, d, f). a) x 100. b) x 100. c) x100. d) x 100. e) x f) x

21

22 Root colonization (%) CO- 1 CO-2 Honey dew Washington Varieties Hyphal --* Arbuscular -- Vesicular S Total Fig. 18: Root colonization of arbuscular mycorrhizal fungi during vegetative stage. Error bar indicate ± 1 SE.

23 (a) CO-1 CO-2 Honey dew Washington Varieties 0 Hyphal M Arbuscular 6 Vesicular 0 Total (b) Root colonization (%) Varieties 0 Hyphal Arbuscular I Vesicular Total Fig.111 Root colonization of arbuscular mycorrhizal fungi during flowering stage - I a) Root colonization in males b) Root colonization in Females Error bar indicate I 1 SE

24 (a) Root colonization (%) Root colonization (%) CO-2 Honey dew Washington Varieties Hyphal --III Arbuscular A Vesicular --A Total (b) CO-1 Washington Hyphal 0 Arbuscular A Vesicular A Total Fig-20 s Root colonization of arbuscular mycorrhizal fungi during flowering stage - II a) Root colonization in males b) Root colonization in Females Error bar indicate t 1 SE.

25 (a) CO-1 CO-2 Honey dew Washington Varieties 4 Hyphal Arbuscular A -- Vesicular A Total (b) Root colonization (%) CO-1 CO-2 Honey dew Washington Varieties Hyphal U Arbuscular A Vesicular A Total Fig.21 : Root colonization of arbuscular mycorrhizal fungi during fruitng stage - I a) Root colonization in males b) Root colonization in Females Error bar indicate ± 1 SE.

26 80 (a) CO-1 CO-2 Honey dew Washington Varieties 10--Hyphal --11 Arbuscular A Vesicular -Al Total Root colonization (%) CO-1 CO-2 Honey dew Washington Varieties * Hypttal U Arbuscular A Vesicular Total : Root colonization of arbuscular mycorrhizal fungi during fruitng stage - IL a) Root colonization in males b) Root colonization in Females Error bar indicate t 1 SE.

27 Vegetative Stage of development ci Root cononization Spore density (b) Flowering I Flowering 11 Fruiting I Fruiting II Stages of development Li Mean total root colonization (%) 12 Mean spore density/100g soil g Flowering I Flowering II Fruiting I Fruiting II Stages of development (c) GI Mean total root colonization (%) La Mean spore density/ 100g soil Fig.23:Mean total root colonization and mean spore density of arbuscular mycorrhizal fungi during different stages of development a) vegetative b) Male plants c) Female plants. Error bar i ndicate t 1 SE.

28 significantly in four varieties of Carica papaya L during vegetative stage. The total root colonization in this stage was ranging from 14.20% to 30.60% whereas, the root length colonized by hyphae, arbuscules and vesicles ranged from 13-20%; 0-10% and 2-6% respectively (Fig.18). The mean total root colonization recorded for the vegetative stage was 24.50% (Fig 23a), whereas the mean values recorded for root length colonized by hyphae, arbuscules and vesicles were 17.15%, 2.45% and 4.30% respectively (Table 51). b) Male plants: In male plants during flowering stage-i, arbuscular (C.D = 3.735; P= 0.05), vesicular (C.D = 4.752; P= 0.05) and total root colonization (C.D = 5.08; P= 0.05) of arbuscular mycorrhizal fungi varied significantly in four varieties of Carica papaya L. however, hyphal colonization did not vary significantly within the varieties. During this stage, hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi ranged from 10-15%, 12-43%, 4-10% and % respectively (Fig. 19 a). In flowering stage-ii, the hyphal (C.D = 3.758; P= 0.05), arbuscular (C.D = 4.549; P= 0.05), vesicular (C. D = 2.701; P= 0.05) and total root colonization (C.D = 6.366; P= 0.05) of arbuscular mycorrhizal fungi also varied significantly in four varieties of Carica papaya L. The hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi in this stage ranged from 15-38%; 18-46%; 6-20% and 47-82% respectively (Fig.20a). The mean total root colonization of arbuscular mycorrhizal fungi (Fig. 23b) and the root 159

29 length colonized by hyphae, arbuscules and vesicles were higher in flowering stage II as compared to flowering stage I (Table 51). Further, the total root colonization of arbuscular mycorrhizal fungi (C. Di = 4.747;.P= 0.05 and C. D2 = 4.040; P= 0.05) and root length colonized by hyphae (C. Di = 3.641; P= 0.05 and C. D2 = 3.264; P= 0.05), arbuscules (C. Di = 3.179; P= 0.05 and C. D2 = 3.657; P= 0.05) and vesicles (C. D i = 3.800; P= 0.05 and (C. D2 = 1.371; P= 0.05) varied significantly among the four varieties during fruiting stage-i and fruiting stage-ii respectively. The hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi during fruiting stage-i ranged from 2-15%; 6-40%; 6-25% and % respectively (Fig. 21a). Whereas the hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi during fruiting stage-ii ranged from 2-19%; 0-16%; 5-35% and % respectively (Fig. 22 a). The mean total root colonization (Fig. 23 b) and mean arbuscular colonization (Table 51) of arbuscular mycorrhizal fungi was higher in fruiting stage I as compared to fruiting stage II. Whereas hyphal and vesicular colonization of arbuscular mycorrhizal fungi exhibited little or no variation during the two stages. c) Female plants: In female plants during flowering stage-i, hyphal (C.D = 4.811; P= 0.05), arbuscular (C.D = 4.005; P= 0.05), vesicular (C.D = 2.494; P= 0.05) and total root colonization (C.D = 4.711; P= 0.05) of arbuscular mycorrhizal fungi varied significantly in four varieties of Carica papaya L. During this stage, hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi ranged from 3-12%, 3-24%, 0-4.6% and 8-40% respectively (Fig.19 b). In flowering stage-ii, the hyphal (C.D = 3.185; P= 160

30 TABLE 51 : Mean values of root length colonized by arbuscular mycorrhizal structures during different developmental stages of Carica papaya L. STAGES OF DEVELOPMENT *ROOT COLONIZATION (%) HYPHAL ARBUSCULAR VESICULAR Vegetative (4.228) 2.45 (2.000) 4.30 (1.067) Flowering Stage I Male (1.810) (5.578) 6.65 (1.628) Female 7.60 (2.502) (4.030) 1.00 (0.958) Flowering Stage II Male (4.502) (5.224) (2.571) Female (3.578) (13.160) (1.58) Fruiting Stage I Male 7.25 (2.705) (8.01) (4.654) Female (3.300) (8.222) (4.520) Fruiting Stage II Male 9.00 (1.967) 5.55 (1.531) (1.628) Female (3.300) 4.75 (8.219) (4.520) *Values presented are mean of five readings; Values in the parenthesis indicates ± 1 SE.

31 also varied significantly in four varieties of Carica papaya L. The hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi in this stage ranged from 18-33%; 20-57%; 10-15% and % respectively (Fig. 20a). The mean total root colonization of arbuscular mycorrhizal fungi (Fig. 23c) and the root length colonized by hyphae, arbuscules and vesicles were higher in flowering stage II as compared to flowering stage I (Table 51). Further, the total root colonization of arbuscular mycorrhizal fungi (C. D 1 = 5.554; P= 0.05 and C. D2 = ; P= 0.05) and root length colonizes by hyphae (C. D1 = 3.341; P= 0.05 and C. D2 = 3.029; P= 0.05), arbuscules (C. D 1 = 3.341; P= 0.05 and C. D2 = 3.349; P= 0.05) and vesicles (C. D1 = 4.651; P= 0.05 and C. D2 = ; P= 0.05) varied significantly among the four varieties during fruiting stage-i and fruiting stage-ii. The hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi during fruiting stage-i ranged from 2-20%; 0-46%; 4-28% and % respectively (Fig. 21b). Whereas the hyphal, arbuscular, vesicular and total root colonization of arbuscular mycorrhizal fungi fruiting stage- II ranged from 8-15%; 2-8%; 12-20% and 22-40% respectively (Fig. 22 b). The mean total root colonization (Fig. 23 c) and mean arbuscular colonization (Table 51) of arbuscular mycorrhizal fungi was higher in fruiting stage I as compared to fruiting stage II. Whereas hyphal and vesicular colonization of arbuscular mycorrhizal fungi exhibited little or no variation during the two stages. 161

32 Acid and Alkaline phosphatase activity- Comparative account of alkaline and acid phosphatase activities in four varieties of Carica papaya L. during different stages of development is recorded in Table 52 and Table 53. Throughout the study period, Washington variety recorded the highest phosphatase (alkaline and acid) activity followed by Honey dew, CO-2 and CO-1 variety (Table 52). Also in the present study, acid phosphatase activity was higher compared to alkaline phosphatase activity in four varieties of Carica papaya L. during different stages of development (Table 52 and Table 53). Both alkaline and acid phosphatase activity varied significantly among the four varieties for any particular stage of development (Table 53). a) Vegetative stage: The alkaline and acid phosphatase actives ranged from n moles of p-npp released / min/ gg protein and n moles of p-npp released / min / gg protein respectively (Table 52). The mean alkaline and mean acid activities recorded during this stage were 5.92 and n moles of p-npp released / min / pg protein respectively (Table 53). b) Male plants: In flowering stage-i, the alkaline phosphatase activity ranged from n moles of p-npp released / min / 14 protein whereas, the acid phosphatase activity ranged from n moles of p-npp released / min / gg protein. In flowering stage II, the alkaline and acid phosphatase activities ranged from n moles of p-npp released / min / gg protein and n moles of p-npp released / min / gg protein (Table 52). The mean alkaline and mean acid activities were higher 162

33 TABLE 52 : Comparative account of phosphatase activity in roots of Carica papaya L. varieties during different stages of development. Stages of development * Varieties of CaHca papaya L. CO-1 CO-2 HONEY DEW WASHINGTON Alkaline Acid Alkaline Acid Alkaline Acid Alkaline Acid Vegetative 1.46 (0.537) 9.62 (2.503) 1.88 (0.638) (5.149) 7.86 (2.698) (3.213) Flowering Stage I Male (2.934) (5.564) (4.662) (10.708) (12.699) (17.064) Female (0.477) (10.708) (1.205) (1.916) (1.994) (2.607) Flowering Stage II Male (0.317) (1.506) (1.686) (6.818) (3.562) (12.52) Female (0.720) (5.537) (0.814) (3.689) (2.217) (9.655) Fruiting Stage I Male , (0.626) (0.891) (0.671) (0.856) (0.525) (1.568) Female (0.571) (1.368) (1.069) (3.439) (0.179) (0.768) Fruiting Stage II Male (0.160) (0.276) (0.139) (0.208) (0.249) (0.247) Female (0.11) (0.186) (0.101) (0.197) (0.263) (0.317) *Values presented are mean of five readings; Values in the parenthesis indicates ± 1SE 8.35 (0.727) (39.327) (29.223) (28.33) (54.323) 5.55 (0.350) 5.55 (0.359) 2.46 (0.158) 1.42 (0.285) (2.616) 767 (65.996) (35.70) (32.019) (24.101) (3.453) (0.834) 6.39 (0.23) (0.724)

34 TABLE 53 : Mean values of alkaline phosphatase and acid phosphatase activities during different developmental stages of Carica papaya L. STAGES OF DEVELOPMENT *PHOSPHATASE ACTIVITY (n moles of p-n PP released/ min 1 pg of protein) Vegetative Mean alkaline phosphatase ' C. D (P= 0,05) Mean acid phosphatase ' C. D (P= 0.05) 5.92 (1.81) (6.26) Flowering Stage I Male (59.34) (142.06) Female (19.461) (30.44) Flowering Stage II Male (26.442) (28.91) Female (70.69) (98.15) Fruiting Stage I Male 7.14 (2.365) (11.92) Female (0.748) (10.769) Fruiting Stage II Male 1.75 (5.501) (2.46) Female 2.48 (0.581) (2.66) *Values presented are mean of ten readings; Values in the parenthesis indicates ± 1 SE. test significant at 0.05 level of probability.

35 in flowering stage-i as compared to flowering stage- II (Table 53).The alkaline phosphatase activities during fruiting stage-i and fruiting stage- II ranged from n moles of p-npp released / min/ lig protein and n moles of p-npp released / min / pg protein. Whereas the acid phosphatase activities ranged p- NPP released / min / lig protein and p- NPP released / min / tig protein (Table 52). The mean alkaline and mean acid activities were higher in fruiting stage-i as compared to fruiting- II (Table 53). c) Female plants: In flowering stage-i, the alkaline phosphatase activity ranged from n moles of p-npp released / min / lig protein whereas, the acid phosphatase activity ranged from n moles of p-npp released / min / lig protein. In flowering stage II, the alkaline and acid phosphatase activities ranged from n moles of p-npp released / min / lig protein and n moles of p-npp released / min / protein (Table 52). The mean alkaline and mean acid activities were higher in flowering stage-ii as compared to flowering stage- I (Table 53). The alkaline phosphates activities during fruiting stage-i and fruting stage-ii ranged from n moles of p-npp released / min/ lig protein and n moles of p-npp released / min / lig protein. Whereas the acid phosphatase activities ranged p- NPP released / min / lig protein and p- NPP released / min / lig protein (Table 52). The mean alkaline and mean acid activities were higher in fruiting stage-i as compared to fruiting stage-ii. 163

36 D Spore density of arbuscular mycorrhizal fungi- Comparative account of spore density of arbuscular mycorrhizal fungi in four varieties of Carica papaya L. during different stages of development is presented in Table 54 and Fig. 23. Unlike root colonization and root phosphatase activity, spore density did not exhibit any definite patterns within the four varieties during different developmental stages of Carica papaya L. Also spore density of arbuscular mycorrhizal fungi recorded a narrow range of fluctuation during the study period as compared to root colonization and phosphatase activity. a) Vegetative stage: Spore density of arbuscular mycorrhizal fungi varied significantly within the four varieties of Carica papaya L. during the vegetative stage (C. D = ; P= 0.05). The highest and the lowest spore density recorded during this period was 20 spores/ 50g soil (C0-1 and Honey Dew) and 8 spores/ 50g soil (Washington) (Table 54). The mean spore density of arbuscular mycorrhizal fungi recorded during vegetative stage was 15.5 spores/ 50g soil (Fig. 23 a). b) Male plants: The spore density of arbuscular mycorrhizal fungi in male plants varied significantly during flowering stage-i (C.D = ; P= 0.05), flowering stage-ii (C. D = ; P= 0.05), fruiting stage-i (C. D = ; P= 0.05) and fruiting stage-ii (C. D = ; P= 0.05) of Carica papaya L. varieties. During flowering stage-i and II, the spore density varied from 10 spores/ 50 g soil (C0-1 and Washington) to 34 spores/ 50 g soil (CO- 2) and 24 spores/ 50 g soil (Honey dew) to 59 spores/50gsoil (C0-1) respectively (Table 54). In fruiting stage-i, maximum spore density of 39 spores/ 50 g soil was recorded in CO -2 variety whereas minimum spore 164

37 density of 17 spores/ 50 g soil was recorded in Washington and CO-1 variety each. In fruiting stage-ii, the spore density ranged from 14 spores/ 50 g soil (C0-1) to 38 spores/ 50 g soil (Honey dew) (Table 54). The mean spore density of arbuscular mycorrhizal fungi in male plants was maximum (30.10 spores/50g soil) during fruiting stage-h and minimum during flowering stage-ii (17.33 spores/50 g soil) (Fig. 23 b). c) Female plants: The spore density of arbuscular mycorrhizal fungi in female plants varied significantly during flowering stage-ii (C. D = ; P= 0.05), fruiting stage-i (C. D = ; P= 0.05) and fruiting stage-ii (C. D = ; P= 0.05) of Carica papaya L. varieties. However spore density of arbuscular mycorrhizal did not vary significantly within the four varieties during flowering stage- I. In this stage, the spore density ranged from 30 spores/ 50 g soil (C0-1 and Honey Dew) to 38 spores/ 50 g soil (Washington) (Table 54). In flowering stage-ii, the highest spore density of arbuscular mycorrhizal fungi was recorded in CO-1 variety (29 spores/ 50 g soil) whereas the lowest spore density was recorded in CO-2 variety (16 spores/ 50g soil) (Table 54). In fruiting stage-i and fruiting stage-ii, the spore density ranged from 10 spores/ 50g soil (Washington) to 33 spores/50g soil (Honey Dew) and 24 spores/ 50 g soil (C0-1 and CO-2) to 46 spores/50g soil (Washington) respectively (Table 54). The mean spore density of arbuscular mycorrhizal fungi in female plants was maximum (33.75 spores/50g soil) during flowering stage-i and minimum during fruiting stage-i (19.09 spores/50 g soil) (Fig. 23c). 165

38 TABLE 54 : Spore densities of arbuscular mycorrhizal fungi during different developmental stages of Carica papaya L. Stages of *Spore density/ 50g soil. development CO-1 CO-2 HONEY DEW WASHINGTON Vegetative (3.000) (1.830) (3.810) 8.00 (0.850) Flowering Stage I Male (0.838) (1.645) (3.278) (1.139) Female (1.466) (2.835) (1.143) (2.671) Flowering Stage II Male (1.370) (1.520) (3.826) (2.764) Female (2.816) (1.000) (1.551) (0.634) Fruiting Stage I Male (1.793) (2.0Q5) (1.582) (1.268) Female (0.896) (1.520) (2.888) (0.838) Fruiting Stage II Male (1.227) (2.414) (0.448) (1.765) Female (1268) (1.183) (1.380) (2.004) *Values presented are mean of five readings; Values in the parenthesis indicates ± 1 SE

39 Relation ship between arbuscular mycorrhizal fungal structures - In the present study, mean total root colonization of arbuscular mycorrhizal fungi exhibited highly significant positive correlation with mean hyphal (r = 0.679; P= 0.01), mean arbuscular (r = 0.894; P= 0.01) and mean vesicular (r = 0.717; P= 0.01) colonization (Table 55). The study also recorded significant positive correlation between mean hyphal and mean vesicular colonization (r = 0.337; P= 0.05) and highly significant correlation between mean hyphal and mean arbuscular colonization (r = 0.429; P= 0.01). Relation ship between root colonization and spore density of arbuscular mycorrhizal fungi No significant correlation was recorded between root colonization parameters and spore density of arbuscular mycorrhizal fungi (Table 55). Relation ship between root colonization and phosphates activity- The study recorded highly significant positive correlation between acid and alkaline phosphatase activity (r = 0.967; P= 0.01). Among the root colonization parameters considered for the study, mean total root colonization recorded significant positive correlation with alkaline (r = 0.410; P= 0.05) and acid phosphatase (r = 0.420; P= 0.05) activity (Table 56). Whereas the mean arbuscular colonization recorded highly significant positive correlation with alkaline (r = 0.531; P= 0.01) and acid phosphatase (r = 0.551; P= 0.01) activity (Table 56). Further, multiple regression analysis was carried out to study the relationship between root colonization and phosphatase activity at each stage. 166

40 TABLE 55 : Correlation coefficient (r) between root colonization and spore density of arbuscular mycorrhizal fungi in Carica papaya L. Parameters Mean root colonization (%) Mean spore density 150g soil Hyphal colonization Arbuscular colonization) Vesicular colonization Total colonization Mean Hyphal colonization (%) Mean arbuscular colonization (%) ** 1.00 Mean vesicular colonization (%) Mean total colonization (%) Mean spore density (%) 0.337* ** 0.894** 0.717** *Correlation significant at 0.05 level of probability. ** Correlation significant at 0.01 level of probability.

41 TABLE 56 : Correlation coefficient (r) between phosphatase activity and root colonization in Carka papaya L. PARAMETERS PHOSPHATASE ACTIVITY (n moles of p-npp released/ min / ig of protein) Mean alkaline phosphatase Mean acid phosphatase Mean alkaline phosphatase (n moles of p-npp released/ min / pg of protein) Mean acid phosphatase (n moles of p-npp released/ min / pg of protein) " 0.967*" 1.00 Mean Hyphal colonization (%) Mean arbuscular colonization (%) 0.531' 0.551' Mean vesicular colonization (%) Mean total colonization (%) 0.410* 0.420* *Correlation significant at 0.05 level of probability. " Correlation significant at 0.01 level of probability.

42 It was observed that during vegetative stage, all the four parameters viz., hyphal, arbuscular, vesicular and total root colonization contribute to both alkaline and acid phosphatase activity. However coefficient of regression was significant only for alkaline phosphates activity (Table 57). During flowering stage-i and flowering stage - II, it was observed that phosphatase activity was significantly influenced by root colonization of arbuscular mycorrhizal fungi in male plants as well as female plants (Table 58 and 59). In male plants, during flowering stage-i, all the four parameters of root colonization significantly influenced the alkaline and acid phosphatase activity (Table 58). In flowering stage II, it was observed that vesicular colonization did not influence phosphatase activity, whereas the remaining three parameters viz., hyphal, arbuscular and total root colonization contributed significantly to phosphatase (alkaline and acid ) activity (Table 59). In female plants, a reverse trend was recorded as compared to male plants. During flowering stage I, vesicular colonization did not influence the phosphatase activity while the other three parameters viz., hyphal, arbuscular and total root colonization significantly to phosphatase activity, whereas in flowering stage II, all the four parameters significantly influenced alkaline as well as acid phosphatase activity (Table 58 and 59). The values of regression coefficient reveal that the fit of the equations are most appropriate. In fruiting stage I, alkaline and acid phosphatase activities were significantly influenced by total root colonization and root length colonized by hyphae, arbuscules and vesicles in male plants as well as female plants of Carica papaya L. (Table 60). However in fruiting stage-ii, all the root 167

43 TABLE 57 : Relationship between root phosphatase activity (Y) and root colonization of arbuscular mycorrhizal fungi (X) in Carica papaya L. during vegetative stage. Phosphatase activity Equations1 Regression coefficient (n moles of p- NPP released! min/ lig protein) (R2) talkaline Y= H 0.53 A V tacid Y= H +1.84A V * *Regression significant at 0.05 level of probability. t Ym = Equation for male plants, YF = Equation for female plants. (IP T= Total root colonization and root length colonized by hyphae (H), arbuscules (A) and Vesicles (V).

44 TABLE 58 : Relationship between root phosphatase activity (Y) and root colonization of arbuscular mycorrhizal fungi (X) in Carica papaya L. during flowering stage I. Phosphatase activity (n moles of p- NPP released/ min/ pg protein) Equationsc Regression coefficient (R2) talkaline Ym= H A V * YF = H+9. 26A * tacid Y M = H A V * YF = H+11.35A+5.21T 0.876* *Regression significant at 0.05 level of probability. t Ym= Equation for male plants, YF = Equation for female plants. T= Total root colonization and root length colonized by hyphae (H), arbuscules (A) and Vesicles (V).

45 TABLE 59 : Relationship between root phosphatase activity (Y) and root colonization of arbuscular mycorrhizal fungi (X) in Carica papaya L. during flowering stage II. Phosphatase activity (n moles of p- NPP released/ min/ pg protein) EquatIonsq Regression coefficient (R2)?Alkaline Y m = H A T 0.903* YF = H A V T 0.732*?Acid Ym= H 0.21 A T 0.757* YF = H A V T 0.726* *Regression significant at 0.05 level of probability. t Ym = Equation for male plants, YF = Equation for female plants. T= Total root colonization and root length colonized by hyphae (H), arbuscules (A) and Vesicles (V).

46 TABLE 60 : Relationship between root phosphatase activity (Y) and root colonization of arbuscular mycorrhizal fungi (X) in Carica papaya L. during fruiting stage I. Phosphatase activity (n moles of p- NPP released/ min/ pg protein) Equationsl Regression coefficient (R2) tatkaline YM = H A V T 0.872* YF = H A V T 0.800* tacid Ym = H A 0.03 V T 0.651* YF = H A V 0.88 T 0.569* *Regression significant at 0.05 level of probability. Ym= Equation for male plants, YF = Equation for female plants. IT= Total root colonization and root length colonized by hyphae (H), arbuscules (A) and Vesicles (V).

47 colonization parameters significantly contributed to only acid phosphatase activity (Table 61) in male and female plants of Carica papaya L. Relation ship between edaphic factors, arbuscular mycorrhizal fungi and root phosphatase activity- In the present study only one of the total nine edaphic factors recorded significant relationship with root colonization and phosphatase activity. It was observed that available phosphorus levels in the rhizosphere soil recorded significant negative correlation with mean arbuscular colonization (r = , P= 0.05) (Table 62) and mean alkaline phosphatase activity (r = ; P= 0.05) (Table 63). Available phosphorus also recorded highly significant correlation with mean acid phosphatase activity (r = ; P= 0.01) (Table 63). However, the study recorded absence of correlation between spore density of arbuscular mycorrhizal fungi and edaphic factors (Table 62). Distribution of arbuscular mycorrhizal fungi during different stages of development All the species of arbuscular mycorrhizal fungi associated with Carica papaya L. varieties during the different stages of growth sporulated in pot cultures. with an exception of two species viz., Glomus coremioides and Glomus sinuosum. Additionally, one species of arbuscular mycorrhizal fungi viz., Glomus macrocarpum was exclusively recovered from pot cultures and was not recorded in the original rhizosphere field samples. This species was recorded in the pot cultures established from rhizosphere soil collected during flowering stage-i (Table 64 and Table 69). 168