Screening and evaluation of indigenous halo-tolerant microbes for salt stress alleviation in celery ( Apium graveolens )

Salinity is one of the major environmental threats which damages growth and productivity of the plants. Microbial assistance in such stressful environment is well recognized. Here in this study, we isolated indigenous microbes and investigated their rescuing potential in celery plants grown under salinity stress. Celery is a widely consumed plant in salads. Plants were cultivated under varying levels (5 & 10% in aqueous solution against control) of salinity in the greenhouse with inocula of two isolated strains of rhizobacteria (RB) which were screened from locally collected soil samples. Data (chlorophylls, carotenoids, anthocyanins, fresh and dry weights of plants, and lengths of root and shoot) were collected and analysed using SPSS. Biochemical isolation of the rhizobacteria was also performed. Plants inoculated with the isolated rhizobacterial strains indicated a statistically significant relief to the stressed plants which resulted in more chlorophylls’ (a, b & total), carotenoid and anthocyanin contents that were at par with control. Post inoculation elongation of root and shoot as well as fresh and dry matter accumulations were enhanced significantly. RB 20 indicated statistically significant relief to the plants compared to RB 10. Bacterial strains screening results showed that strains RB 6 & RB 20 proved their positive relieving strengths in the tests of indole synthesis, siderophore production, phosphorus solubilization, casein hydrolysis, catalase activity, citrate biosynthesis, gelatinase biosynthesis, H 2 O 2 production, motility test, osmotic regulation potential and starch hydrolysis. Hence, these indigenous microbes might be helpful in assisting celery plants grown under salinity conditions.


Introduction
Salinity is one of the major environmental concerns that can affect crop productivity and quality and can cause a reduction in the cultivated land area (Shahbaz and Ashraf, 2013). Salt stress is a very important abiotic stress which has affected one third of the world's irrigated land making it unsuitable for cultivation. Plants growth processes like seed germination, seedling development and vigor, flowering, fruit setting etc are adversely affected by salinity stress. Consequently, salinity not only lowers the quality of the produce but also reduces yield (Rabhi et al., 2012). Plants do react to this stress in a number of ways i.e. stopping the entry of salts (at the whole plant or cellular level), taking extra water from soil, decreasing the concentration of the salts in the cytoplasm etc (Teakle and Tyerman, 2010).
Celery is an outstanding verdant vegetable and is specially grown in China, Pakistan, North America, Iran, Europe and India. Its oil also has helpful applications in pharmaceutical and fragrance industry. The seed of celery has properties like a diuretic, calming and carminative (Marongiu et al., 2013). Celery is a plant with wide uses (Wang et al., 2010).
Salinity has a negative influence on microbial activities going on in the rhizosphere like reduction of microbial growth, community structure change etc (Laderio et al., 2012). However, microbes can tolerate salinity by employing strategies like assembling osmolytes, altering their community structure etc. (Wichern et al., 2006). Soil, plants and microbes strongly interact through soil environment. The non-pathogenic and beneficial rhizospheric microbes referred to as plant growth-promoting bacteria (PGPB) rescue the plants through certain direct and indirect mechanisms (Ramadoss et al., 2013).
The nitrogen fixation, phosphate solubilization and the production of phytohormones and 1aminocyclopropane-1-carboxylate (ACC) deaminase are direct mechanisms to enhance plant growth. Whereas, indirect mechanisms include production of antagonistic compounds such as hydrolytic enzymes, siderophores and variety of antibiotics (Suárez-Moreno et al., 2012;Costa et al., 2014).
Several studies have highlighted the role of PGPB in the mitigation of salt stress and occurrence of indigenous microbes further provides a strong base camp to better fight the salinity stress (Costa et al., 2014;Lubna et al., 2018). Henceforth, present study was designed to explore the indigenous microbiota which can ease out salinity stress in celery.

Isolation and inoculum preparation of halotolerant bacterial strains
Two most outstanding bacterial strains were isolated at the University of Punjab Lahore from soil samples taken from saline land patches of the Varsity Agricultural Farm randomly. Briefly, Soil was mixed and diluted (8X) with double sterilized water by vertexing. Further 10X dilutions were made and aliquots were shifted onto nutrient agar plates. Purified and dissimilar colonies were further cultured in LB media in flasks which were then centrifuged to obtain the pellets. The pellets were washed with sterilized distilled water and diluted to concentrations of 10 7-8 cells/mL by taking OD of 1.0 at 600nm. After the isolation of bacterial strains, the isolates were tested for stress alleviation traits mentioned in table 5 and two strains (RB 6 & RB 20) with maximum potential were utilized in further studies (Damodaran et al., 2013). Biochemical isolation data of the rest strains are not shown.
Celery seeds were sterilized with sodium hypochlorite (1% for 15 minutes followed by 3 to 4 washings with distilled water). Nursery was raised, and 5 seedlings were sown into each pot containing 1 kg sterilized soil (Table 1) and left for 2 weeks to get established. Plants were supplied with distilled water till establishment. Then, the abovementioned treatments were applied to the pots randomly using 50mL of each of the treatment solutions (salt solution & aqueous bacterial solution with adjusted concentration). Same amount of distilled sterilized water was given to the control. Plants were kept watering with salt/control solutions on daily basis.
Harvesting of the plants was done at 30 days post inoculation. The plant growth parameters including shoot length, root length, fresh and dry biomasses were recorded. There were total five replicates.

Estimation of chlorophylls, anthocyanin and total carotenoids
The estimations of Chlorophyll 'a', 'b', total chlorophyll, anthocyanin and carotenoid were done by using the methods as described by Arnon (1949) and Lichtenthaler and Wellburn (1983), respectively.

Data analysis
Data obtained were analysed using ANOVA with the help of SPSS (V. 10) and means were separated by using Duncan's multiple range test at 5% level.

Screening of bacterial strains for plant growth promoting traits
Impact of PGPB strains on growth parameters of plants A significant impact of both the bacterial strains was seen on growth and vigour of A. graveolens plants. An increase of shoot length by 13% and 21% was recorded after the inoculation of strains RB6 and RB20, respectively, under 10% salinity stress conditions compared with control.
Likewise, 26% and 38% increase in root length was noticed post inoculation of microbial isolates RB6 and RB20 in comparison with control under higher salinity level (10% salinity stress), accordingly (Table 2). Similarly, fresh and dry biomasses of the plants were significantly higher over the introduction of RB20 under low level of salinity stress (5%) compared with both the control and RB6, however, both the bacterial isolates were statistically at par at 10% salinity conditions (Table 3).

Impact of PGPB strains on chlorophylls, carotenoids and anthocyanin contents of the plants
Photosynthetic apparatus (chlorophyll a, chlorophyll b and total chlorophyll), carotenoids and anthocyanin production were enhanced upon furnishing of PGPB as compared to control in both the salinity stress levels.
Further, strain RB20 showed significantly better results than RB6 (Table 4). When compared at 5% salinity level, the percent increases in chlorophyll 'a,' chlorophyll 'b,' total    (Table 4). The assistance provided by the microbial isolates of both the types (RB6 & RB20) in achieving the higher syntheses of chlorophylls, carotenoids and anthocyanin was also statistically comparable to the control plants grown with distilled water applications at 5% salinity level (Table 4). As a whole, the assistance provided by strain RB20 in synthesis and maintenance of chlorophylls, carotenoids and anthocyanin was significantly higher than strain RB6 (Table 4).

Biochemical traits screening of the PGPB strains
A comparative depiction of the overall scenario of the biochemical traits of the strains is given in Table 5. Results exhibited that strain RB20 performed significantly better than strain RB6 in IAA synthesis, siderophore production than strain RB6 whereas RB6 failed to show phosphate solubilisation contrary to RB 20 (Table 5). For citrate utilization test, both the strains showed positive influence, however, RB6 expressed a significantly better result as compared to strain RB20 (Table 5). Motility test confirmed the positive involvement of both the strains but RB6 was found superior to strain RB20 (Table 5). Biosynthesis of indole acetic acid was less significant in RB6 than in RB20. Osmotic contents regulation by both the strains was statistically equal except for hydrolysis of starch where a negative result was obtained under RB20 strain application and it was lowered under higher salinity level in both of the strains' applications (Table 5). Similar confirmations could also be seen in Figure 1.

Discussion
PGPB have been reported in relieving plants under salinity stress by improving the overall growth performance of the plants ( Damodaran et al., 2013;Lubna et al., 2018). Plant roots explore the soil and provide nutrients and other benefits to the plants. Hence, any impediment in root expansion also disturbs rest of the plant functions including shoot growth. But the presence of plant growth promoting bacteria in the rhizosphere can alleviate this stress and enable the plant to continue its normal functioning (Tank and Saraf, 2010;Ramadoss et al., 2013;Lubna et al., 2018). Our results also confirm that the presence of excessive amount of salts in the soil not only affects the root system elongation but also shoot growth of the celery plants. However, induction of the microbial isolates benefited the plants and restored their overall growth to a larger extent (Table 2). Similarly, salinity adversely affects the photosynthetic pigments of the plants which in turn make plant growth and development stunted (Rabhi et al., 2012;Sang-Mo et al., 2014). Application of PGPB improves the integrity and biosynthesis of chlorophylls (Tank and Saraf, 2010;Sang-Mo et al., 2014). Table 4 clearly indicates the enhanced synthesis of chlorophylls, carotenoids and anthocyanins after the inoculations were provided to the experimental pots. Upregulated biosynthesis of photosynthetic apparatus lead to increased biomass accumulation by celery plants in our study.
Above mentioned relief to the plants is an outcome of the ability of the microbes to support higher productions of the biochemicals involved in rescue activities (Table 5). Better performance by plants under RB20 strain application was probably due to its higher ability to support various  (Table 5). Improved osmotic regulation under salinity stress was noticed upon introduction of isolates to the pots in our experiment ( Figure  1 & Table 5). Indoel acetic acid, siderophore release, phosphorus solubilization, casein hydrolysis, catalase activity, citrate consumption, gelatinase liquefaction, hydrogen sulphide production, osmotic regulation all are the biochemical/traits associated with relief functions in plants under stressful salinity environments (Nadeem et al., 2012;Ribeiro and Cardoso, 2012;Suárez-Moreno et al., 2012;Damodaran et al., 2013;Geetha et al., 2014;Lubna et al., 2018).

Conclusion
A significant enhancement of growth parameters of plants like shoot length, root length, plant fresh biomass and dry biomass upon treatment with PGPB alone in comparison with control. A similar boost was seen when PGPB were applied under salinity conditions. All this was achieved as a result of relief provided to the plants by the microbial inoculations applied. Hence, indigenous microbiota may also be employed in enhancing growth of celery plants under saline conditions.