Sustainable agriculture and horticulture are currently an ongoing research effort for reaching communities current food demand without understanding the future requirement and development.  Quality farming is needed for the maintenance of soil fertility; use of the wide application of manmade agrochemicals (e.g chemical fertilizer) has been a notable contributor to environmental pollution. This research article aims to analyze the potential of Rhodopseudomonas strains a purple non-sulphur bacteria, which promotes plant growth. When used as a commercialized bio-fertilizer Rhodopseudomonas strains is examined based on two main features of sustainability, including the effects on plant growth, environmental impact, and easy production. The productiveness is dependent on the improvement of plant growth through the secretion of extracellular metabolites, resistance to abiotic stresses, bioremediation of heavy metals, and mitigation of greenhouse gas emissions. This article suggests the vitally important roles of Rhodopseudomonas strains as an effective bio-fertilizer in Agriculture and Horticulture. However, the amount of production and application earned more attention. The potential substrates ranging from various waste streams and formulation methods for Rhodopseudomonas strains production are summarized to discuss environmental and economic stability.

Rhodopseudomonas strains, Bacterial biomass, Bio-fertilizer, Seed germination, Agriculture and Horticulture.

Modern farming techniques are very costly and helpful in increasing food and agriculture demand, mostly to the natural environment, such as loss of soil fertility and environmental pollution.  Problems like high mineral fertilization can be decreased by cost effective use of greener biofertilizers.  The plant growth promoting rhizobacteria (bacteria) (PGPR/PGPB) is widely used as biofertilizers have been reported due to their important effects to promote plant growth and yield (Basu et al., 2021). Plant growth is promoted by free living microorganisms.  To increase worldwide crop production there is usage of soil microbes; thus, producing a new bacterial genus as a novel bio-fertilizer to increase plant growth and yield is highly desirable. Rhodopseudomonas palustris (R. palustris) is a free-living bacterium species under the genus Rhodopseudomonas, phylum Proteobacteria.  For the biosynthesis of energy this bacteria uses various biotic and abiotic factors. Rhodopseudomonas palustris is one of the purple non-sulphur bacteria (PNSB). The four modes of Metabolism are chemoautotrophic, photoautotrophic, chemoheterotrophic, and photoheterotrophic, increasing versatility and flexibility, this is widely used in  biotechnological applications because of its multifaceted modes, including a promising bio-fertilizer to be utilized in agriculture. Some beneficial functions of R. palustris include (i) nitrogen (N) fixation (Wong et al., 2014), (ii) heavy metal remediation (Batool et al., 2017) and (iii) methane (CH4) emission mitigation in saline paddy fields (Kantha et al., 2015).  Indole-3-acetic acid (IAA) (Wong et al., 2014) and 5-aminolevulinic acid (ALA) are the plant growth promoters produced by the R. palustris. They also absorb sodium ions with the help of exopolymeric  substances. Which help in stimulation of plant growth and improved hostility to environmental stresses. Another beneficial feature of R. palustris is that it can be prepared in a low-cost effective culture medium facilitating high production (Lo et al., 2020). These features make R. palustris acceptable as a biofertilizer and biocontrol agent for numerous agricultural applications, although an overview on R. palustris multi-functional traits is still lacking. Research has demonstrated that inoculating plants with PGPR as bio-fertilizers can be an effective strategy to increase crop growth. (Backer et al., 2018) have highly reviewed and created the roadmap to commercialize PGPR as biofertilizers. Basu et al. (2021) also have discussed about different aspects of PGPR as biofertilizers, including the beneficial applications, mechanisms of actions and commercialization pathways. Specific PGPR strains have also been studied and covered in different aspects, including Bacillus spp. and their role in plant growth development and stress reduction (Radhakrishnan et al., 2017), the impact of bacteria (Azospirillum species), in agricultural and environmental applications (Cassán et al., 2020), and the different possible operational mechanisms of rhizobia (Jaiswal et al., 2021) were identified. Most of  the highlighted PGPR are from various microbial taxa, with a lack of significance on PNSB to showcase and exploit  as biofertilizers. The inclusion of PNSB like R. palustris as beneficial bio-fertilizers is often failed to see, probably due to the no proper  commercialization or limited application for growth promotion of crops, compared with other bacteria. Recent work by Sakarika et al. (2020) leads to a pathway to purple non-sulphur bacteria, providing a detailed explanation on the use for plant growth, environmental aspects, and preliminary cost-effectiveness analysis. The way of transitioning from research to application has also been discussed focused on the shelf-life issues and application methods. The discussion by Sakarika et al. (2020) is from a relatively high perspective, covering all the phototrophic microorganisms. Our study serves as an updated work that shows the benefits of a specific strain, mainly R. palustris, as promising bio-fertilizers based on three main points of sustainability, including the effects on plant growth, environmental impact, and easy production. The value of R. palustris in agriculture is reviewed and discussed, from their benefits on plant growth to synthesize as low-cost commercial agricultural inputs to promote stable agriculture. The commercialization and application of PGPR as a biofertilizer, such as Pseudomonas spp. and Bacillus spp. among many rhizobacteria, has become a main component of sustainable agriculture practices in many countries (Mustafa et al., 2019).  EmFarma Plus commercialized photosynthetic bacteria  by a manufacturer called Probiotics Polska (Mącik et al., 2020) in Europe. Nun Kaew et al. (2014) have demonstrated the potential of R. palustris as a plant growth-stimulating bacterium at a lower cost than commercial ALA. The successful development of an R. palustris formulation is mainly dependent on using sustainable materials and application methods, such as utilizing horticultural oil as a potential additive for the liquid-based production of R. palustris (Lee et al., 2016). Lo et al. (2020) have developed a low-cost medium to facilitate large-scale production of R. palustris. These findings suggest that R. palustris has the ability to be develop  a safe, cost-effective and easy-to-process microbial formulation that would facilitate its practical use in the field at a wide scale. Despite previous studies they have successfully determined the stable R. palustris formulations using either liquids or solids as carrier materials or efforts to achieve low-cost production, the commercialization of R. palustris inoculant is relatively new and still has a long way to go. Timmusk et al. (2017) presented the challenges and procedures for the commercial synthesis of PGPR/PGPB products. Backer et al. (2018) have proposed ten steps to develop and commercialize PGPR-based inoculants, while Basu et al. (2021) have discussed the industrial production pathways and constraints in developing PGPR bio-fertilizers. Recent work by Sakarika et al. (2019) has explained the roadmap for research and valorization of PNSB products used for plant production. Although these reviews have extensively discussed the plan of action or space to commercialize PGPR- or PNSB-based biofertilizers, the naming and application features are not widely discussed. The commercialization strategy stops  as there is  no correct quality check available for bio-fertilizers at the moment (Basu et al., 2021). For the application of biofertilizers on crops farmers requires correctly labeled information. The quality of bio fertilizers is hard to understand  as there synthesizing process is different from chemical fertilizers (Malusà et al., 2016). The Potential of Rhodopseudomonas Palustris as a Bio-Fertiliser for Sustainable Agriculture Sabki et al. (2021).  Establishing quality control guidelines for biofertilizers is important to avoid failure in the fields. A highly produced  quality standard can help to inform the inoculation efficiency of bio- fertilizers and gain confidence among consumers that leads to creating a more demand in the fertilizers market.

A. In Vivo – plastic tray experiments

(i) Sample preparation of Capsicum annuum.L. Capsicum annuum (Green Chilli) crop is one of the important crop in Telangana State of India belong to Nightshade family. It is consumed in the form of spices in different curries and pickles used in homemade items. The present research was conducted with short duration of Capsicum annuum (green chilli) crop. The main objective is to assess the impact of Capsicum annuum on morphology, growth and yield parameters with soil amendment of selected strains of RM01, RM02 and RM03 bacterium at the time of sowing. Capsicum annuum (Green Chilli) seeds were shown in measurement units plastic pots with 16 cm height and 12 cm radius at the top filled with two kg of soil. The sowing was done during the first week of March 2022 in a Green House at Department of Botany, University College of Science, Osmania University, Hyderabad, Telangana, India. The maximum and minimum temperatures recorded during the experimental period rang from 20.5°C to 32.8°C. The crop was maintained stress free conditions by irrigating at regular intervals.

Preparation of Rhodopseudomonas sp. biomass:  Rhodopseudomonas sp. RM01, RM02 and RM03 were grown in modified Biebl and Pfennig’s (1981) medium. Three bacterium were grown in a ten litter capacity cultured bottle and incubated under 3,000 lux illumination intensity with 28±2oC temperature in anaerobic conditions. Indigenous Rhodopseudomonas sp. self-flocculated and on the fifth day the culture were taken for preparation of experimental biofertilizer. The fine powder of soil was thoroughly mixed with ratio of 1: 3 (Biomass: Soil).

(ii) Treatment of Capsicum annuum.L with PNSB culture. Fifty milliliters  of  logarithmically grown cultures of all three strains, such as  Rps. palustris RM01, Rps. rhenobacensis RM02, Rps. palustris RM03 centrifuged at 6000 rpm and pellet were washed with distilled water and again centrifuged to get pellet. 50 ml of distilled water were added to this pellet and resultant suspension was added to soil of each pots at time of sowing chilli plants (Arunasri, 2004). Biofertilizer studies were carried out using horticulture crop i.e., Capsicum annuum (green chillies) for the current study. The efficiency of three strains were tested by observing the morphological and biomass parameters like plant height (cm),number of leaves, leaf area (cm2/pl), root length (cm), root volume (ml/pl), dry weight(g/pl) of leaf, stem, root and total biomass were tested with all the three strains of PNSB. The observation were recorded at 18th day, the experiment were conducted with three bacterial strains (Rhodopseudomonas palustris RM01, Rhodopseudomonas rhenobacensis RM02, Rhodopseudomonas palustris RM03). Four Plastic trays were used for seeding each tray contains fifty cavities of depth 3.8 cm, size 54 × 26 cm × 4.5(L×W×H) pack of one tray. Garden soil was taken into the plastic trays.  For the seed germination water sprinkled were twice day. The sprouts were observed from tray. The sprouted grains are separated carefully. Out of four plastic trays, one tray used for Rhodopseudomonas palustris RM01 biomass (1g/kg soil) second tray used for Rhodopseudomonas rhenobacensis RM02 biomass (1g/kg soil)  3rd tray used for Rhodopseudomonas palastris RM03 biomass (1g/kg soil) and last one for control each of  which contains fifty seed capacity trays. The seed germination were recorded at regular intervals and   plants from all the three treatments were harvested on zero day to sixth  month and parameters such as plant root length (cm),  root volume (ml), shoot length (cm), leaf length (cm), leaf width (cm), Total height of the plant, number of leaves, number of branches, number of clusters, number of pods, plant dry biomass (g/pl), seed weight (g/pl) and husk (seed coat) weight were recorded.

The germinated seeds were removed carefully at seedling stage and washed out. The shoot and root measurements of the plants were calculated. For studies of biofertilizer efficacy of phototsynthesic bacteria, the soil with  photosynthetic bacteria dry biomass powder were taken separately at the dose of 3grams (RM01, RM02 and RM03) bacterial biomass per kg of soil separately. The shoot length, leaf length & leaf width, root length and the number of  leaves were measured at 8th day,10th day, 12th day,16th day  and 18th  day and height of chilies plants were measured at the 30th day , 60th day, 90th day, 120th day,  150th day and 180th day. The growth parameters like plant height, leaves of the plant, shoot and root length; fresh and dry weight of shoot and root were measured at 180day. Studies of yield number of fruits, average size of fruits, weight of seed in each fruits and total number of seeds/fruits were also measured at 180days. 

Table 1: Isolation of Bacteria and production of large scale biomass (biofertilizer).

Table 2: Field screening of most effective bacterial biomass and production of crop yield.

Among the various biotechnological applications of Purple Non-Sulfur Bacteria, its usage as biofertilizer is gaining importance in recent times. The results obtained from field experiments with soil amendment of PNSB biomass to a crop plant were presented. Pot experiments were carried out to quantify the effect of three PNSB strains as soil amendment on the seed germination of Capsicum annuum L and plant growth under experimental conditions. In horticulture and agriculture used two types of chillies and from that one is green chilli which are used  in daily cooking as a vegetable  and second one is normal chillies used for a commercial use in pickles and industrial purpose Current study were conducted on green chilli crop (Capsicum annuum). In the market so many bacterial based biofertilizer are available but phototrophic bacteria as pigment based biofertilizer, has shaded therefore we have selected for study as  biofertilizer. We were selected green chillies used for study to check the effect of crop yield by using PNSB as a biofertilizer.

A. Preparation of bacterial biomass for biofertilizer studies

The present investigation were carried out to study the effect of bacterial biomass on chilli crop, using as biofertilizer compared with control plants (without biomass biofertilizer). The biomass of three PNSB strains (RM01, RM02 and RM03) were taken individually, 3 grams of biomass were added to 100 grams of normal garden soil and mixed well and chilli seeds were sowed in tissue culture poly thin tray, which were filled with soil with bacterial biomass.  

Fig. 1. Bacterial culture of three PNSB strains.

(i) Seed germination of Capsicum annuum L. Seed germination and plant growth conditions of green chillis of selected model green chilli seeds, were widely studied in the literature, to investigate the individual and the combined effects of control RM01, RM02 and RM03 biomass biofertilizer treatment on the rate of seed germination and plant growth. 

Fifty chilli seeds were sowed in three plastic trays separately, each plastic tray containing fifty seeds single treatment were used for all the experiments carried out  under the same conditions to confirm the reproducibility. Seed samples were divided into four groups as shown as follow: 

Sample-1: Plastic tray containing fifty seeds used as control (without any treatment biomass). 

Sample-2: Plastic tray containing fifty seeds used as test (treatment RM01 biomass).

Sample-3: Plastic tray containing fifty seeds used as test (treatment RM02 biomass).

Sample-4: Plastic tray containing fifty seeds used as test (treatment RM03 biomass).

All the trays were watered with 10 ml of tap water every day at 24hr interval and placed in green house chamber at room temperature and relative humidity which were located in Department of Botany, University College of Science, Osmania University, Hyderabad, Telangana state, India. The number of seed germination were observed and recorded every day. The germination rate were calculated at 0th , 8th, 10th, 12th  and 18th  days depending on the nature of the seeds. For the growth studies of plants, control (untreated) and treated seeds were planted in green house nursery pots.

(ii) Rate of germination of Capsicum annuum L. The number of seeds sprouted divided by the number of total seeds germinated, then multiplied by hundred. The equation is to calculate the germination percentage is:

Formula : % of Seed  Germination = No. of Germinated Seeds/Total Seeds × 100

The rate of germination provides a measure of the time course of seed germination by hundred.

B. Effect of RM01, RM02 and RM03 biomass on seed germination of (Capsicum annuum)

In this section the effect of seed germination were evaluated & compared with control. The germination of chilli seeds have been examined every 0th Day, 8th Day, 10th Day, 12th  Day and 18th days along with control. Control seeds, RM01, RM03 at 12th day and RM02 at 9th day examined the seed germination (Fig. 2 and Table 3). 

Fig. 2. Influence of biomass on seed germination of plants. 

As compared to control plants, Rhodopseudomonas palustris RM01 and Rhodopseudomonas rhenobacensis (RM02) and Rhodopseudomonas palustris RM03 biomass has enhanced the seeds germination rate and the seedling growth. As seen in (Table 3). After 18th day the percentage (%) of seed germination were observed as 82% (control), 88 (%) RM01, 92(%) RM02 & 84 (%) RM03 respectively. In addition, the RM01 and RM03 biofertilizer showed increased rate of seed germination and also improved the seedling growth than control.

Table 3: Effect of three bacterial biomass of RM01, RM02 and RM03 on seed germination of Capsicum annuum.L (green chillies).

Fig. 3. The growth of root length, shoot length in (cm) and Root volume in (ml) growth from 0th -18th day seedling in control.

Fig. 4. The growth of root length (cm), shoot length in (cm) and root volume in (ml) chillies 0th -18th day of seedling in soil treated with RM01 biomass.

Fig. 5. The growth of root length (cm), shoot length in (cm) and root volume in (ml) chillies 0th -18th day of seedling in RM02 treatment with PNSB biomass

Fig. 6. The growth of root length (cm), shoot length in (cm) and root valume in (ml) chillies 0th -18th day of seedling in soil treated with RM03 biomass.

After cultivation of chilli plants, the percentage of seed germination (%) observed in which the growth were more in RM01, RM02 and RM03 when compared to Control. Therefore, in order to understand the long term combined effect of treated chilli, % of seed germination of chilli plants were calculated at 0th . 8th , 10th , 12th  and 18th day .The seed germination of RM01, RM03 biomass treated plants showed less seed germination when compared with RM02 biomass The maximum seed germination percentage was observed on 18th day of sowing the seeds. In our observation the percentage of seed germination inhibition in all three PNSBs were reported as 12% in Rps. palustris  RM01,  8%  in  Rps. rhenobacensis RM02, 16%  Rps. Palustris RM03, 18% in control without biofertilizer  respectively. The maximum seed germination were observed on 9th day in case of RM02 where as the RM01, RM03 and control showed on 12th day.

C. Effect of bacterial biomass on various parameters of Capsicum annuum L

The present investigation were carried out to study the effect of bacterial biomass on chilli crop. The bacterial biomass of RM01, RM02 and RM03 inoculated in plants showed increase in the growth of chilli plants when compared with control plants. All the parameters like morphological and bio-chemical parameters were increased in soil which is inoculated with bacteria biomass. The present study were well correlated with the previous reports by Gaur and Agarwadi (1989). Where they studied the combined and dual inoculations of A. brasilense and Pseudomonas strain in sorghum plant which increased in root length, nitrogenase activity and dry matter, seed yield as compared to single inoculation of both organisms and control plants (Fig. 7- 26,  Table 4).

(i) Effect of Bacterial Biomass on the Morphological Parameters of Chilli Leaves.

Fig. 7. Effect of bacterial biomass RM01, RM02 and RM03 on the growth of number of leaves.

Fig. 8. Effect of bacterial biomass RM01, RM02 and RM03 on the length of  leaves.

Fig. 9. Effect of bacterial biomass RM01, RM02 and RM03 on the leaf width.

Fig. 10. Measurement of  Leaves length  (cm) and width (cm) in 30th days.

Fig. 11. Effect of  three bacterial biomass on shoot length, leaves length, root length, root volume and total height of the plants at 60th  days.

Fig. 12. Effect of three Bacterial biomass on shoot length, root length and root volume, total height of plant compared with control in chillies in 90th days.

Fig. 13. Effect of three bacterial biomass on shoot length, root length and total height of chillies plants compared with control in on 120 days, 150th day and 180th day.

(ii) Effect of Bacterial Biomass on the Morphological Parameters on Shoot Length, Root Length and Root Volume of Chilli Plant

Fig. 14. Effect of RM01, RM02 and RM03 bacterial biomass on the shoot length.

Fig. 15. Effect of bacterial biomass RM01, RM02 and RM03 on  root length.

Fig. 16. Effect of bacterial biomass RM01, RM02 and RM03 on root volume.

(iii) Effect of Bacterial Biomass on the Morphological Parameters on Number of Flowers and Total Length of Chilli Plant

Fig. 17. Effect of bacterial biomass RM01, RM02 and RM03 on Number of flowers.

Fig. 18. Effect of bacterial biomass RM01, RM02 and RM03 Total length of shoot, root, chillies)

Fig. 19. The yield of  Capsicum annuum L collected at 120th days.

Fig. 20. Effect of bacterial biomass (biofertilizer) RM01, RM02 and RM03 on growth of fruit and seeds in chilli plants.

Fig. 21. Effect of bacterial biomass (biofertilizer) RM01, RM02 and RM03 on pigment ( Fruit colour) and growth of chilli.

Table 4: Comparative morphological analysis of control & soil treated with bacterial biomass of RM01, RM02 and RM03 effect on growth of Capsicum annuum L.

Fig. 22.  Effect of bacterial biomass on the fruit size of Capsicum annuum L.

Fig. 23. Effect of bacterial biomass on the fruit width of Capsicum annuum L.

Fig. 24.  Effect of bacterial biomass on the fruit weight of Capsicum annuum L.

Fig. 25.  Effect of bacterial biomass on the seed weight of Capsicum annuum L.

Fig. 26. Effect of bacterial biomass on the number of seeds in fruit in Capsicum annuum L.

M. Manjula, K. Rama  and B. Rajani  (2024). Effect of Rhodopseudomonas Strains Biomass on Morphological Parameters of Seed Germination of Capsicum annuum L. Biological Forum – An International Journal, 16(8): 129-139.