Role of Pellets and Capsules of Acacia nilotica and Sapindus mukorossi in Combination of Seed Bio-Priming with Microbial Antagonists in the Supression of Root Infecting Pathogenic Fungi and Promotion of Crop Plants

Acacia nilotica (L.) Willd. ex Del. is also known as Gum Arabic tree, Babul, Egyptian thorn, or Prickly. Acacia is a multipurpose tree and play a great role in fixation of nitrogen [1]. Species of Acacia contains secondary metabolites including amines and alkaloids, cyanogenic glycosides, cyclitols, fatty acids and seed oils, fluoroacetate, gums, nonprotein amino acids, terpenes (including essential oils, diterpenes, phytosterol and triterpene genins and saponins), hydrolyzable tannins, flavonoids and condensed tannins [2]. A. nilotica also possess antimicrobial activity against some micro-organisms [3]. Mashram has observed the antimicrobial activity of Acacia nilotica, against microorganisms like, B. subtilis, S. aureus and E. coli in vitro and found that the extracts of bark and leaf showed inhibition zone between 7.5-16 and 8-15.5 mm respectively and most effective against E. coli. and B. subtilis. recorded antifungal activity of methanolic extracts and aqueous extract of A. nilotica with percentage inhibition ranging from 34.27 ± 1.45 to 93.35 ± 1.99 [4]. Similarly Sapindus mukorossi (L.) is well known for its folk medicinal values. Recently many of the pharmacological actions of this plant have been explored which includes the antimicrobial cytoxoic molluscicidal insecticidal and fungicidal [5-9].The leaves are used in the baths to relieve joint pain and the roots are used in the treatment of gout and rheumatism. Since ancient times S. mukorossi has been used as a detergent for shawls and silks. The fruit of S. mukorossi was utilised by Indian jewellers for restoring the brightness of tarnished ornaments made of gold, silver and other precious metals [10]. Dawar et al. [11] studied that leaves, stem, bark and fruit powder of Eucalyptus sp., has ability to reduce the infection of root infecting fungi viz., Fusarium sp., R.solani and M. phaseolina.


Introduction
Acacia nilotica (L.) Willd. ex Del. is also known as Gum Arabic tree, Babul, Egyptian thorn, or Prickly. Acacia is a multipurpose tree and play a great role in fixation of nitrogen [1]. Species of Acacia contains secondary metabolites including amines and alkaloids, cyanogenic glycosides, cyclitols, fatty acids and seed oils, fluoroacetate, gums, nonprotein amino acids, terpenes (including essential oils, diterpenes, phytosterol and triterpene genins and saponins), hydrolyzable tannins, flavonoids and condensed tannins [2]. A. nilotica also possess antimicrobial activity against some micro-organisms [3]. Mashram has observed the antimicrobial activity of Acacia nilotica, against microorganisms like, B. subtilis, S. aureus and E. coli in vitro and found that the extracts of bark and leaf showed inhibition zone between 7.5-16 and 8-15.5 mm respectively and most effective against E. coli. and B. subtilis. recorded antifungal activity of methanolic extracts and aqueous extract of A. nilotica with percentage inhibition ranging from 34.27 ± 1.45 to 93.35 ± 1.99 [4]. Similarly Sapindus mukorossi (L.) is well known for its folk medicinal values. Recently many of the pharmacological actions of this plant have been explored which includes the antimicrobial cytoxoic molluscicidal insecticidal and fungicidal [5][6][7][8][9].The leaves are used in the baths to relieve joint pain and the roots are used in the treatment of gout and rheumatism. Since ancient times S. mukorossi has been used as a detergent for shawls and silks. The fruit of S. mukorossi was utilised by Indian jewellers for restoring the brightness of tarnished ornaments made of gold, silver and other precious metals [10]. Dawar et al. [11] studied that leaves, stem, bark and fruit powder of Eucalyptus sp., has ability to reduce the infection of root infecting fungi viz., Fusarium sp., R.solani and M. phaseolina.
Pelleting and encapsulation of plant parts gaining importance in recent times stated that pyrophyllite mixed Avecenia marina plant parts pellets (leaves and stem powder mixed separately with pyrophillite) had played a marked role in the elevation of growth parameters as well as in the reduction of soil borne root rot fungi like F. oxysporum, M. phaseolina and R. solani on cowpea and bringal plants [12]. Ghaffar, 1995 observed prominenet reduction in M. phaseolina infection on chickpea and mungbean plants when pellets of sodium alginate were mixed in the soil @ 1 and 10 pellets inside the plastic pots.
Walker and Connick [13] used alginate type pellets in formulations of microbiological and chemical herbicides. According to, Tariq and Dawar [14] encapsulation of halophytic plant parts powder such as stem and leaves powder @ five capsules per pot prominently decreased the incidence of root-infecting pathogenic fungi on okra and mungbean plants.
Apart from pelleting and encapsulation techniques, bio-priming of seeds with beneficial micro-organisms is also gaining importance in the control of many plant pathogens as another alternative to synthetic fungicides in present times. Bio-priming of seeds with antagonistic microbes are capable of colonizing the rhizosphere by potentially providing the advantages to the plant beyond the seedling emergence stage. Using antagonistic microorganisms was such an approach which was used increasingly on a commercial scale both in field and green house crops. Antagonistic micro-organisms like Trichoderma species are important biological control agents (BCAs) of many soil borne plant pathogens [15]. Different mechanisms have been used by Trichoderma in order to control plant pathogens which include competition for nutrients and habitat, mycoparasitism, release of antibiotics and fungal cell wall breaking enzymes [15][16][17]. Besides fungal antagonistic microbes, beneficial bacterial isolates have also been used for seed biopriming. These beneficial bacterial micro-organisms contain antifungal and good plant growth promoting attributes. Bacterial strains in soil that have tremendous effects on plant growth and health are commonly known as plant growth promoting rhizobacteria (PGPR). PGPR enhance plant growth indirectly or directly with the biocontrol of pathogens, production of plant hormones or improvement of plant nutritional status [18]. Present research work was therefore carried out on role of pellets and capsules of A. nilotica and S. mukorossi in combination of seed bio-priming with microbial antagonists in the suppression of root infecting pathogenic fungi and promotion of crop plants.

Collection of plants and antagonistic agents
A. nilotica and S. mukorossi leaves parts were collected from Campus of University of Karachi, air dried separately and finely powdered in an electric grinder. Cultures of Rhizobium meliloti and Trichoderma harzianum were obtained from the Karachi University Culture Collection (KUCC).

Preparation of pellets and filling of capsules
A. nilotica and S. mukorossi pellets were prepared with the help of research method of Tariq and Dawar [12]. Similarly, A. nilotica and S. mukorossi pellets were prepared with the help of multiple pellet sampler of equal size and weight (1 g pellet containing 0.5 g pyrophyllite and 0.5 g leaves powder). These pellets were air dried under laminar air flow chamber. Leaves powder of A. nilotica and S. mukorossi were filled in empty capsules (0.5 g in each capsule).

Preparation of spore/cell suspension and bio-priming of seeds
Spore/cell suspension of T. harzainum and R. melilotii were prepared in distilled sterilized water and seeds were bio-primed in these suspensions separately for about 10 minutes and then these seeds were air dried for sowing.

Pots setup under screen house
Plastic pots containing 300 g of soil were placed under screen house in Department of Botany, University of Karachi under randomized block design. These experimental pots containing (a) amendment of A. nilotica and S. mukorossi pellets in the soil separately in which non treated seeds were sown (b) T. harzianum and R. melilotii primed seeds were sown in the soil separately in non-amended soil (c) combined effect of both bioprimed seeds and pellets amended soil (d) only pyrophyllite pellets were mixed in the soil which served as control no. 1 (e) 0.5 g of A. nilotica and S. mukorossi leaves powder were filled in empty capsules separately and mixed in the soil containing non treated seeds (f) combined effect of both capsules and bioprimed seeds (g) empty capsules mixed in the soil which regarded as control no. 2. These experimental pots were kept under screen house for about 30 days and for maintaining the moisture content watered regularly. After 30 days the plants were uprooted for the observation of growth parameters and their roots were washed in running tap water for estimation of roots colonization by root infecting fungi.

Statistical Analysis
Data obtained from the experiment were analysed with the help of ANOVA (analysis of variance) and LSD (least significant difference) test at P=0.05 and DMRT (Duncan's multiple range test) in order to compare treatment means, using STATISCA computer software.

Results
There was significant (p<0.001) enhancement in growth parameters of peanut when A. nilotica pellets (pyrophyllite and leaves powder @ 50:50 ratio) in combination with bio-priming of seeds with T. harzianum spore suspension was used as compared to the control in which only pyrophyllite pellets was amended in the soil (Figure 1). Similarly combine effect of A. nilotica capsules (filled with 0.5 g leaves powder) and bio-priming of peanut seeds with T. harzianum spore suspension gave significant increase in root length, shoot length, root weight and shoot weight and prominent decrease was also noticed in the colonization of Fusarium sp (p<0.001), R. solani (p<0.01) and M. phaseolina (p<0.5) as compared to the control (empty shells of capsules) ( Figure 1). In chickpea, root length, shoot length, root weight and shoot weight increased significantly (p<0.001) when A. nilotica pellets and capsules were amended in the soil and seeds were bio-primed with T. harzianum, whereas, M. phaseolina reduced significantly (p<0.5) in comparision with both the controls ( Figure 2). Combine impact of A. nilotica (pellets) and S. mukorossi (capsules) along with T. harzianum bio-primed seeds significantly (p<0.001) elevated the growth parameters of okra and significant suppression in root infecting fungal pathogens like R. solani (p<0.001) and Fusarium sp (p<0.001)   was also observed ( Figure 3). In sunflower, combined application of (A. nilotica pellets and biopriming of seeds with T. harzianum) and (encapsulation of S. mukorossi and T. harzianum primed seeds) gave significant (p<0.001) health and vigour to growth parameters such as root length, shoot length, root weight and shoot weight and significant decrease in root infecting fungi such as Fusarium sp (p<0.001), R. solani (p<0.001) and M. phaseolina (p<0.5) was also recorded ( Figure  4). Of all the treatments and amendments, it was observed that pellets and capsules of A. nilotica, and S. mukorossi in combination with biopriming of seeds with T. harzianum spore suspension was found to be most effective for the promotion of growth and suppression of root infecting pathogenic fungi like Macrophomina phaseolina, Rhizoctonia solani and Fusarium sp on leguminous and non-leguminous crops.

Discussion
In the present research, encapsulation and pyrophyllite mixed pellets of A. nilotica and S. mukorossi alongwith T. harzainum primed seeds played a marked role in the enhancement of growth parameters as well as in the reduction of fungal pathogens like M. phaseolina, R. solani and Fusarium sp of leguminous and non-leguminous crops. Significant (p<0.001) enhancement in growth parameters of peanut was noticed when A. nilotica pellets (pyrophyllite and leaves powder @ 50:50 ratio) in combination with bio-priming of seeds with T. harzianum spore suspension was used. Tariq and Dawar [12] used formulations of A. marina plant parts pellets with pyrophyllite @ 50:50 ratio and observed that there was prominent increase in growth parameters of leguminous and non-leguminous plants and root infecting fungi like M. phaseolina, R. solani and Fusarium sp was also reduced significantly .Our results showed that in chickpea, root length, shoot length, root weight and shoot weight increased significantly with the combined effect of A. nilotica pellets, capsules and T. harzianum primed seeds whereas, M. phaseolina reduced significantly. Similarly, when mixed alginate pellets in the soil, there was significant suppression in the colonization of M. phaseolina on mung bean and chickpea plants [19]. According to our results, combined application of A. nilotica (pellets) and S. mukorossi (capsules) along with T. harzianum bio-primed seeds significantly elevated the growth parameters of okra and significant suppression in root infecting fungal pathogens like R. solani and Fusarium sp was also observed. Tariq and Dawar [20] investigate the effect of mangrove plant parts filled capsules and pellets and recorded that formulations of mangrove combined parts powder and pellets when amended in the soil found to releases compounds which are nematicidal in nature and reduced the activity of Meloidogyne javanica on okra and mungbean plants and thus increase plant growth and crop yield.  Besides pelleting and encapsulation reports, several studies on biopriming of seeds with beneficial micro-organisms have shown that seed bio-priming with microbial antagonists played a prominent role in the health and vigour of seedlings as well as in the reduction of many soil borne diseases. Rafi and Dawar [21] studied the effects of bio-priming on leguminous and non-leguminous crops at different time intervals and concluded that growth parameters markedly improved when seeds were bio-primed with microbial antagonists like Trichoderma harzianum and Rhizobium meliloti for 10 minutes whereas, root infecting fungal pathogens reduced significantly when seeds were bio-primed with T. harzianum, Bacillus sp and R. meliloti conidial/cell suspensions for 5, 10 and 20 minutes time interval .Present investigation found that in sunflower, combined application of (A. nilotica pellets and biopriming of seeds with T. harzianum) and (encapsulation of S. mukorossi and T. harzianum primed seeds) gave maximum growth parameters such as root length, shoot length, root weight and shoot weight and significant decrease in root infecting fungi was also recorded. Harman [22] observed that strain T-22 of Trichoderma harzianum causes plant to have more extensive root systems and not only suppressed the diseases but also improve the plant metabolism. Present research investigation clearly suggests that pelleting and capsulation of A. nilotica, and S. mukorossi in combination with biopriming of seeds with T. harzianum spore suspension was found to be most effective for the promotion of growth and suppression of root infecting pathogenic fungi like Macrophomina phaseolina, Rhizoctonia solani and Fusarium sp on all the tested plants.