Phosphate solubilizing endophytic actinomycetes from mangrove plants Rhizophora mucronata and Sonneratia caseolaris and its effect on Seedling vigour

 

Sunita C. Mesta, R. Onkarappa*, Meghana S.H, Manu H.M, Kavana S, Mohamed Talib

Department of Studies and Research in Microbiology, Sahyadri Science College (Autonomous), Kuvempu University, Shimoga-577203, Karnataka, India.

 

ABSTRACT:

The aim of the present study was to isolate phosphate solubilizing endophytic actinomycetes from two mangrove plants Rhizophora mucronata and Sonneratia caseolaris, to estimate phosphate solubilization quantitatively and to check its efficiency on Seedling vigour. The plant parts were surface sterilized and were plated on Oat meal agar supplemented with 10% sea water and a total of 11 endophytic actinomycetes were isolated. The isolates were screened for Phosphate solubilization using Pikovskaya’s agar medium. The potent isolates were subjected for fermentation and the broth culture was subjected for quantitative estimation of phosphate solubilization  by Johnson and Koening method. The effect of extracts of endophytic actinomycetes was also evaluated by seed germination and seedling vigour test. Of the total 11 isolates, Isolate RO 4, RO 7, RO 9, RO 10 and RO 11 demonstrated Phosphate solubilizing activity in the form of halo zone around the colonies and were selected for further studies. The broth cultures showed high amount of phosphate release compared to the standard. Highest amount of phosphate was recorded for Isolate RO 11 about 1410µg/ml. Isolate RO 7 and RO 11 showed the highest effect on Seedling vigour index. The results of the present study reveal that phosphate solubilizing endophytic actinomycetes can be used as a source of biofertilizer.

 

 

 

INTRODUCTION:

Endophytic actinomycetes are the microbes that reside in healthy tissues of living plants, without causing clinically detectable symptoms of disease, without having any negative impact on host plant ^[1,2,3]. Actinomycetes are the filamentous gram positive microbes with high G+C content primarily saprophytic and contribute in breakdown of complex biopolymers such as hemicelluloses, pectin, lignocelluloses, keratin and chitin ^[4].

 

The association of actinomycetes with plants is found to confer many advantages such as the production of growth promoting metabolites, insect and pest repellents, anti-microbials, extracellular enzymes, phytohormones and siderophores protectors in stress conditions and many more. They also help in phosphate solubilization and plant protection against abiotic and biotic stresses ^[5,6]. Endophytic actinomycetes are considered to be potential biocontrol agents as they can colonize the interior of the host plant avoiding competition by other microbes in the soil^{[7, 8]}.

 

Phosphorus is the second important key element after nitrogen as a mineral nutrient in terms of quantitative plant requirements ^[9,10]. Although abundant in soils, in both organic and inorganic forms, it’s availability is restricted as it occurs mostly in insoluble forms. It plays significant role in increasing root ramification and strength thereby imparting vitality and disease resistance capacity of plant. It also helps in seed formation and in early maturation of crops like cereals and legumes ^[11]. Poor availability or deficiency of Phosphorus drastically reduces plant size and growth. In this regard Phosphorus- solubilizing microorganisms (PSM) have been seen as best eco-friendly means for Phosphorus nutrition of crop ^[12]. Microbes solubilize the bound phosphate and rock phosphate into simple phosphate. They secrete organic acids such as formic acid, lactic acid, succinic acid, propionic acid which change the soil pH and solubilize the bound phosphate present in the soil ^[13]. Vigour testing is an important component of seed testing because its more sensitive test than germination, and because of loss of vigour may be noted much earlier than loss of germination. Vigour tests are designed to mimic poor seedling conditions to find out how the seed lot will perform under stress. It’s the exact opposite of a germination test where seed is grown under optimum conditions. In a vigour test, the seed is introduced to a stressful environment unfavorable to seedling development. This environment can be cool, cold or warm, or a combination of either high humidity and high temperatures, or heavy moisture at low temperatures. If the seed lacks vigour, one or more of these created stressors will suppress seedling growth but, if the seed is vigorous, it will withstand one or all of these stressors and grow as if it were on stimulants^[14].

 

Mangrove ecosystem is the most productive ecosystem diversified with variety of microbes ^[15,16]. Reports are available confirming occurrence of phosphate solubilizing bacteria and fungi from mangroves. Studies on phosphate solubilizing endophytic actinomycetes are less documented. The present work was an attempt to screen the endophytic actinomycetes isolates obtained from Mangrove plants for phosphate solubilization and quantitative estimation and to check the efficiency of the potent isolates on seed germination and seedling vigour.

 

MATERIALS AND METHODS:

Collection of Mangrove plants:

Two Mangrove plants were collected from Cortalim, Goa, India during January 2017 and were authenticated by Department of Botany, Sahyadri Science College (Autonomous), Shimoga. The plant parts included leaves, bark, roots, stem and pods. The plants were collected in sterilized polythene bags and were stored in the Laboratory at 4^o C until use.

 

Isolation of Endophytic Actinomycetes:

The sterilized explants were cut into small pieces and placed on petriplates containing oat meal agar medium (Himedia laboratories, India) (5 segments of explants per petridish) and incubated at 30^o C for 7-14 days. After the incubation period, the individual colonies with characteristic actinomycete morphology emerging out from the plant tissue were isolated. Streak plate method was used to purify the endophytic actinomycetes cultures on Starch Casein Agar media and were preserved in refridgerator at 4^oC and used for further investigation.

 

Characterization of Endophytic Actinomycetes:

 The isolates were subjected for morphological characterization (colour of aerial and substrate mycelium, pigment production), biochemical tests (Starch hydrolysis, Casein hydrolysis, KOH solubility test, Citrate Utilization test), microscopic (cover slip method) and staining (Gram’s staining)^[17,18,19].

 

Screening of Phosphate solubilizing Endophytic actinomycetes:

Pikovskayas agar media (supplemented with 10% sea water) was prepared and poured into sterile petriplates and allowed to solidify. After solidification, all the endophytic actinomycetes isolates were inoculated on Pikovskaya agar medium by point inoculation and were incubated at 30°C for seven days. After the incubation period all the plates were observed for halo zone formation around the colony which is presumptive confirmation of phosphate solubilization^{[10, 13]}.

 

Quantitative estimation of Phosphate:

The potent isolates that showed phosphate solubilization were selected and inoculated into 250ml conical flasks containing Pikovskays broth and incubated at 30^oC for 10 days. The uninoculated broth was kept as control. After incubation the contents of the flask including the control were filtered through Whatmann No 1 filter paper. The resulting clear filtrate was analyzed for quantitative amount of soluble phosphate. 1ml of aliquote of neat culture filtrate 0.5ml of Bartons reagent was added and the volume was made upto 10ml using Distilled water and were incubated at room temperature for 10-30 min. After the incubation period, the resultant colour developed was read at 430nm in a colorimeter. The amount of released phosphate was expressed in µg/ml by ploting a graph against a standard curve^{[20, 21]}.

 

Effect of Phosphate solubilising endophytic Actinomycetes on Seed germination and seedling vigour by Paper Towel Method:

The paper towel (Rolled towel) method was employed to know the effect of potent phosphate solubilising endophytic actinomycetes on seed quality parameters of maize seeds i.e. to carry out germination and vigour tests ^[14,22]. Two varieties of maize seeds were selected for this study i.e. Pioneer (small seeds) and Srikar (big seeds). Hundred randomly selected maize seeds of both varieties were surface sterilized with 0.1% mercuric chloride for 2min followed by 70% alcohol and repeated washing with distilled water three to four times. One set of surface sterilized seeds were treated with pikovskayas broth and placed on five long lanes on moistened and sterilized blotting paper sheets (two layers below and one layer above). Ten seeds were arranged per lane at equidistance and were noted control seeds. Another set of seeds were dipped in respective culture filtrate for about half an hour before subjecting them to trails. After that the seeds were placed on the moistened and sterilized blotter sheets in the similar manner and were noted as Treated seeds. The blotter paper containing seeds were rolled carefully to avoid any excess pressure on seeds. The paper towels were placed in a sterile polythene cover and were kept in an upright position for 7days. This experiment was carried out under sterile conditions, at temperatures ranging between 25–29°C, and 12 h light – 12 h darkness.

 

All the seedlings were counted on the 7^th day of incubation period and the percentage of germination was calculated. To find out the seedling vigour, the root length (cm) of each seedling was measured. The same seedlings were used for the measurement of the shoot length (cm). The experiment was conducted twice to record the average values.

Percentage of Seed germination was calculated by the formula

 

% of SG = Total number of seeds germinated x 100

       Total number of seeds placed

 

Vigour index was calculated according to the formula

 

VI = SG × (SL + RL)

 

Where VI – vigour index, SG – seed germination (%), SL – mean shoot length (cm),

RL – mean radical length (cm), (SL + RL) – seedling length.

 

RESULTS:

Selection of plants: The two mangrove plants Rhizophora mucronata commonly called loop root mangrove (Rhizophoraceae) and Sonneratia caseolaris commonly called crabapple mangrove or mangrove apple (Lythraceae) were selected for our study.

 

Isolation of endophytic actinomycetes:

A total of 11 endophytic actinomycetes were isolated from both plants. 7 isolates were obtained from Rhizophora mucronata (5 from stem, 1 from bark and 1 from leaf). 4 isolates were obtained from Sonneratia caseolaris (3 from stem, 1 from leaf and no isolates from bark). All the isolates were obtained on Oat meal Agar media and were subcultured on Starch Casein Nitrate Agar media and were preserved in refrigerator at 4⁰C. The number of endophytic actinomycetes isolated are represented in Table 1.

 

Table 1: Endophytic actinomycetes isolates

 

Fig 1: Representative forms of Endophytic actinomycetes isolates

 

 

 

Characterization of endophytic actinomycetes isolates

Morphological characteristics:

The morophological characteristics of isolates revealed a wide range of aerial and substrate mycelial colours such as white, grey, light grey, creamy white. None of the isolates produced diffusible pigments. The spore arrrangement studies revealed a diverse morphological characters with varied spore colour, diffrerent arrays of spore arrangement that varied from Flexibilis, Retinaculum aparatum – open loop, hooks, simple spira and compact spira. The morphological and microscopic characterization of endophytic actinomycetes is represented in Table 2; Fig 2.

 

Table 2: Morphological characterization of actinomycetes

ISOLATES	AERIAL MYCELIUM	SUBSTRATE MYCELIUM	SPORE ARRANGEMENT	TENTATIVE GENERA
RO 1	Creamy white	Light brown	Micromonospora	Micromonospora
RO 2	Grey	Grey	Flexibilis	Streptomyces Sp
RO 3	Cream	Brown	Flexibilis	Streptomyces Sp
RO 4	White	White	Retinaculum aparatum- Hook	Streptomyces Sp
RO 5	Greish white	Brown	Flexibilis	Streptomyces Sp
RO 6	Peech	Light brown	Flexibilis	Streptomyces Sp
RO 7	Grey	Creamy white	Spira	Streptomyces Sp
RO 8	Grey	Creamy	Open spiral	Streptomyces Sp
RO 9	Grey	Creamy white	Retinaculum aparatum- spiral	Streptomyces Sp
RO 10	Dark grey	White	Hook	Streptomyces Sp
RO 11	Light grey	Creamy white	Spiral	Streptomyces Sp

 

 

Fig 2: Representative forms of spore morphology of endophytic actinomycetes

 

Staining and Biochemical Characteristics:

All the 11(100%) isolates were found to be Gram positive and were negative for KOH solubility test and positive for Casein hydrolysis. 7 (63.63%) isolates were positive for starch hydrolysis, 6 (54.54%) isolates were positive for citrate utilization test. The staining and biochemical characteristics of the isolates are represented in Table 3.

 

Table 3: Staining and Biochemical Characteristics of the isolates

Isolate No	Grams Staining	KOH solubility Test	Casein Hydrolysis	Citrate utilization Test	Starch Hydrolysis Test
RO-1	+	-	+	-	-
RO-2	+	-	++	-	-
RO-3	+	-	+	-	-
RO-4	+	-	+++	+	+++
RO-5	+	-	+++	+++	-
RO-6	+	-	+	-	+++
RO-7	+	-	++	+++	+
RO-8	+	-	+	+++	+
RO-9	+	-	+	+++	+
RO-10	+	-	+++	-	+
RO-11	+	-	++	+++	+

 

Screening of endophytic actinomycetes for phosphate solubilization:

The production of halo zones is due to ability of microbes to solubilize insoluble mineral phosphates like calcium phosphate present in the medium. Among the 11 actinomycetes isolates, RO 1, RO 2, RO 3, RO 5, RO 6, RO 8 did not show phosphate activity. The maximum zone of clearance was found with RO 4 (25mm), RO 7(16mm), RO 11(14mm), RO 9 (13mm) and RO 10 (11mm). The preliminary screening of endophytic actinomycetes for phosphate solubilization are represented in Table 4; Fig 3.

 

Table 4: Preliminary screening of PO₄ solubilization

Isolates	Zone of Hydrolysis	Isolates	Zone of Hydrolysis
RO 1	No zone	RO 7	16mm
RO 2	No zone	RO 8	No zone
RO 3	No zone	RO 9	13mm
RO 4	25mm	RO 10	11mm
RO 5	No zone	RO 11	14mm
RO 6	No zone

 

Fig 3: Phosphate solubility test of endophytic actinomycetes

 

 

Quantitative estimation of Phosphate:

Phosphate solubilization in Pikovskaya’s broth medium for the potent isolates was quantitatively analysed. After the incubation period it was observed that isolate RO 11 solubilized and released phosphate about 1410µg/ml followed by RO 7 about 1380µg/ml, RO 10 about 1130µg/ml, RO 9 about 1000 µg/ml and RO 4 about 900µg/ml respectively. The values are represented in Table 5.

 

 

 

Table 5: Quantitative measurement of phosphate solubilization in Culture medium

“Where RO 4, RO 7, RO 9, RO 10, RO 11 = culture filtrate”

 

Effect of Phosphate solubilising endophytic Actinomycetes on Seed germination and seedling vigour by Paper Towel Method:

The seeds treated with culture filtrate had a significant influence on the shoot and root production compared to the seeds treated with broth (control). 100% germination was observed in both varieties of seeds treated with culture filtrate. In control 70% germination was observed for Pioneer (small seeds) variety and 86% germination for Srikar (Big seeds) variety. Seedling vigour index was found to be more in case of seeds treated with culture filtrate of Isolate RO 7 and RO 11 followed by RO 9 and RO 10. The seedling vigour test values are represented in Table 6.

 

Table 6: Seedling vigour test

Parameters	CONTROL		TREATED (Culture filtrate)
	Pioneer	Srikar	Pioneer (small seeds)				Srikar (Big seeds)
			RO 7	RO 9	RO 10	RO 11	RO 7	RO 9	RO 10	RO 11
Total seeds	100	100	100	100	100	100	100	100	100	100
Number of germinated seeds	70	86	100	100	100	100	100	100	100	100
Number of infected seeds	06	02	-	-	-	-	-	-	-	-
Germination percentage	70%	86%	100%	100%	100%	100%	100%	100%	100%	100%
Infection percentage	6%	2%	0	0	0	0	0	0	0	0
Average root length	136	141.5	134	168	136	129.8	196	189.2	192	199.95
Average shoot length	170	277.9	256	184.5	170	230	481.2	394.5	352.75	450.45
Seed vigour index	21,420	36,068.4	39,000	35,250	30,600	35,980	67,720	58,370	54,475	65,040

 

 

Fig 5: Seed germination and seedling vigour test

Photographs showing difference in growth of seed germination and seedling vigour.

 

 

 

DISCUSSION:

Endophytic actinomycetes have worldwide distribution and adapt themselves to varied extreme environments. These microorganisms are known to synthesize numerous novel compounds that can be exploited in agricultural, pharmaceutical and other industries ^{[2, 23-25]}. In the present study a total of 11 endophytic actinomycetes were obtained from root, bark, stem of mangrove plants Rhizophora mucronata and Sonneratia caseolaris. Similar studies have shown that diverse group of endophytic actinomycetes are present in mangrove plants and its environment i.e  rhizosphere soil of mangrove plant Avicennia marina ^[24], medicinal plants ^[3,8,26], mangrove forests ^[27], ethnomedicinal plants ^[28]. Most of the actinomycetes were identified as Streptomyces species by morphological characteristics, which was consistent with the other reports from different hosts.

 

Phosphorus solubilization is a complex process, which is influenced by different factor such as nutritional richness and physiological and growth pattern of the microbial form^[11]. The microorganisms present in the soil convert the unavailable form of phosphorus in available form through their metabolic activities by excreting organic acids. This leads to the decrease in pH of soil and brings about solubilization. Microbial solubilization of mineral phosphate might be either due to acidification of external medium or the production of chelating substances that increase phophate solubilization. Out of 11 isolates screened for phosphate solubilization, 5 (45.44%) isolates showed solubilization of phosphate by formation of halo zone around the colony. These results coincide with that of ^[29](sediment from estuary) ^[13](soil sample from coastal region), who reported the occurrence and distribution of phosphate solubilizing microorganisms in marine environments and Pikovskaya’s medium is specific medium for isolation of phosphate solubilizers ^{[5, 9, 10]}.

 

Quantitative estimation of phosphate solubilization was done by Johnson and Koening method. The amount of phosphate solubilized and released in the broth culture of Isolate RO 11 and RO 7 were more compared to the standard phosphate. Similar results have reported by ^[10] from two strains of Aspergillus niger group out of 14 different genera of fungal organisms screened. Studies by ^[30] reported that amount of phosphate released from rock phosphate was higher in the presence of actinomycetes strains indicating the important contribution of rock phosphate solubilization.

 

Seed treatment often improves the germination standard by controlling of seed surface flora. Vigour testing is very sophisticated and reveals the true quality of the seed. It is one of the most important tools and can be used as a guide to avoid planting into unfavorable conditions ^[14]. The seeds treated with culture filtrate of isolate RO 7 and RO 11 had a significant influence on shoot and root production compared to the control. Similar type of studies on seed germination and seedling vigour was carried out on different seeds by ^[31](Bengal gram, peas), ^[32](rice), ^[33] (sorghum seeds), ^[34](Italian ryegrass, cocksfoot, timothy). In all the cases the seeds treated with the cultures showed maximum germination.

 

CONCLUSION:

The endophytic actinomycetes species plays an important role in solubilizing phosphate in marine ecosystem and increases the soluble phosphate concentration thereby enhancing the productivity of the aquatic system. Isolates RO 7 and RO 11 were found to be best in solubilizing phosphate and promoting seed germination and seedling vigour compared to other isolates. In future these promising strains may be utilized as biofertilizers so as to increase the agricultural yields and in reclamation of barren places.

ACKNOWLEDGEMENT:

The authors are thankful to the Principal, Sahyadri Science College (Autonomous) for facilities and moral support for carrying out the project.

 

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Received on 07.10.2017       Modified on 18.11.2017

Accepted on 06.12.2017      © RJPT All right reserved