Inoculation effect of Azospirillum brasilense on basil grown under aquaponics production system Jonathan S. Mangmang, Rosalind Deaker & Gordon Rogers
Organic Agriculture Official journal of The International Society of Organic Agriculture Research ISSN 1879-4238 Org. Agr. DOI 10.1007/s13165-015-0115-5
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Author's personal copy Org. Agr. DOI 10.1007/s13165-015-0115-5
Inoculation effect of Azospirillum brasilense on basil grown under aquaponics production system Jonathan S. Mangmang & Rosalind Deaker & Gordon Rogers
Received: 10 March 2015 / Accepted: 24 March 2015 # Springer Science+Business Media Dordrecht 2015
Abstract The potential of Azospirillum brasilense to enhance basil growth under aquaponics system was evaluated using the three strains, i.e., Sp7, Sp7-S, and Sp245. Basil seedlings were inoculated with strains of A. brasilense a week after sowing and before transplanting. Impacts of inoculation on some parameters associated with plant growth, and physiological and metabolic activities were evaluated at seedling stage and at harvest of marketable plant size from aquaponics system. Likewise, impacts of inoculation on the existing bacterial communities were assessed at the end of each stage. At seedling stage, inoculated seedlings produced longer (90 %) roots and taller (19 %) plants with more developed (25 %) and bigger (61 %) leaves. As a result, seedling fresh and dry biomasses were increased by 79 and 44 %, respectively, particularly for those seedlings inoculated with Sp7 and Sp245. Some of the plant metabolic activities were altered by inoculation such as elevated levels of enzyme peroxidase activity and total phosphorus content. In aquaponics, plants previously inoculated with the strains also showed superior growth performance. For instance, basil leaf area, fresh herbage yield, and root weight were increased by inoculation up to 27, 11 and 11 %, respectively. Inoculation also enhanced peroxidase activity (73 %), endogenous plant indole-3-acetic acid (IAA) (27 %) and protein contents (20 %) particularly for those plants inoculated with Sp7 J. S. Mangmang (*) : R. Deaker : G. Rogers Faculty of Agriculture and Environment, The University of Sydney, Australian Technology Park, Eveleigh, NSW 2015, Australia e-mail:
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and Sp7-S. Furthermore, Azospirillum inoculation brought no significant disturbance to the composition of indigenous bacterial communities present in the root rhizosphere of the seedlings and aquaponics-grown basil. Thus, this plant growth-promoting rhizobacteria (PGPR) could be an important resource in aquaponics farming to further enhance plant growth and more importantly to increase crop productivity. Keywords Aquaponics . Azospirillum brasilense . PGPR . Recycling . Vegetable
Introduction Conventional agriculture is increasingly dependent on chemical inputs, ground water for irrigation, and land for cultivation. While the use of agrochemicals and the massive agriculture intensification undoubtedly augments crop productivity, their continuous application and land conversion have resulted to unexpected environmental consequences and altered biogeochemical cycles. Suitable crop lands have been gradually encroached by urbanization, which, in turn, reduces the potential land for food production. In this regard, hydroponics could be an option to overcome this challenge. It requires less land and water with high production capacity. This technique also enables growers to have a better control of the environmental factors that affect production and can enable them to grow variety of crops. However, hydroponics is highly dependent on dissolved nutrients from processed chemical fertilizers to sustain production.
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Wastewater from this system, if not recycled, can pose threat to the environment. Hence, an alternative costeffective and more environmental friendly technology is vital to address the abovementioned issues. An emerging food production technique that combines aquaculture and hydroponic cultivation has been considered as one promising alternative to modern agriculture. This integrated system is termed Baquaponics^ that uses wastewater from aquaculture in a sustainable manner through biofiltration. This production system mimics the natural functioning of an ecosystem which carries a promise at a time when natural resources like water and land resources are becoming scarce, and to provide the production of safe and healthy foods, and enhance the local economy. There is a great potential for this sustainable system in highly urbanized areas and in many developing countries where not enough vegetable and protein sources for consumption due to droughts and poor farming practices (Roy et al. 2013). In aquaponics, plants take up the nutrients generated from fish and uneaten fish foods after microbial breakdown. Fish food and waste generally provide most of the nutrients required by plants (Rakocy et al. 2007). However, a few studies have shown that plants grown under this sort of system periodically display symptoms of mineral deficiencies (Adler 1998; Graber and Junge 2009). This could be caused either by insufficient nutrients or inefficient utilization of available nutrients. One way that could address this is to use beneficial microbes, e.g., plant growth-promoting rhizobacteria (PGPR), which are known to improve nutrient utilization and uptake by plants. PGPR are a group rhizospheric bacteria that can colonize plant roots and exert beneficial effects on the host plant when present in ample population. The genera Azospirillum, Bacillus, Burkholderia, Rhizobia, and Pseudomonas are some of the most studied and documented PGPR that have been found to contribute numerous beneficial effects on plants (Saharan and Nehra 2011). Earlier studies have shown that these PGPR can stimulate growth and increase productivity of many important agronomic crops in various conditions. Azospirillum spp. have been studied largely in cereal species and a few other crops. Apart from being a general colonizer, they are considered as versatile PGPR due to numerous plant growth mechanisms. Azospirillum is well known for its ability to produce phytohormones and also to fix atmospheric nitrogen, mineralize nutrients, sequester iron, alleviate biotic and abiotic stresses, survive under harsh condition, and
favor beneficial association of other microflora (Bashan and de-Bashan 2010). They have been found to improve germination, growth, and yield of some crops. However, in some cases, the results were not consistent due to differences on factors such as inoculum concentration, environmental condition, PGPR strains, inoculation method, cultivar, soil type, and substrate condition (Nowak 1998; Zahir et al. 2003). While there may be numerous studies done with Azospirillum inoculation on different crop plants, there has been no reports done to evaluate the effect of this genus on crops grown under aquaponics system. Likewise, studies with basil (Ocimum basilicum L.) are widespread worldwide, but cultivation of this crop inoculated with beneficial microbes under aquaponics system has not been reported in the past, despite the popularity of this culinary herb for fresh and dry leaf usages, essential oils, and seeds. Therefore, this study was conducted to evaluate the effect of Azospirillum brasilense on basil grown under aquaponics system.
Materials and methods Inoculum preparation and inoculation Strains of A. brasilense (i.e., Sp7, Sp7-S, and Sp245) were provided by Dr. Rosalind Deaker, University of Sydney. Each strain was sourced from a pure culture stored with glycerol at −80 °C. Strains were grown in standard nutrient agar and broth. More details of the procedure can be found in the report of Mangmang et al. (2015). Prior to inoculation, the production of indole-3-acetic acid (IAA) in the culture supernatant by A. brasilense strains was measured using a spectrophotometer at 535 nm. One milliliter aliquot of the culture supernatant was mixed vigorously with 4 ml of Salkowski’s reagent (150 ml 98 % H2SO4, 250 ml distilled water, and 7.5 ml 0.5 M FeCl3 ·6H2O), and the mixture was allowed to stand in the dark at room temperature for 20 min (Dobbelaere et al. 1999; Patten and Glick 2002). Absorbance was measured in triplicate, and the concentrations of IAA were determined from the standard curve. The average amount of IAA produced by the strains from two inoculation times was 5.88 ug ml−1. The strains were inoculated at average concentration of 8.65 log colony-forming unit (cfu)ml−1 at first and fourth week after seeding by drenching 5 ml bacterial suspension per seedling pot.
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Seedling production and transplanting A commercial rounded paper pot with a dimension of 80 mm diameter and 75 mm depth filled with a proprietary organic compost plug (Choice Seedlings Proprietary Limited, Werombi, NSW, Australia) was used to raise basil seedlings. Sowing was done by an auto seeder machine (Transplant systems Ltd., Auckland, New Zealand) with approximately 20–30 seeds per pot. Seeds and seedlings were germinated and raised, respectively, in a controlled greenhouse in a commercial nursery of Choice Seedling, Werombi, NSW, Australia (33° 58′ 48″ S, 150° 33′ 59″ E). One week from seedling emergence, thinning was done leaving at least 12 reasonably distant similar size seedlings per pot. All nursery care and management practices (e.g., programmed watering, fertilization, and climate control) of the seedlings were done by the company until ready for transplanting at 28 days from sowing. Seedlings in pots with the organic compost potting medium were directly transplanted into the hydroponic gully at the aquaponics commercial facility of Urban Ecological System Australia (UESA Pty. Ltd.), Cobbitty, Camden, NSW, Australia (33° 00′ 55″ S, 150° 40′ 24″ E). Aquaponics setup This multi-million dollar fish and plant facility is owned by UESA. This aquaponics facility is a dual level stateof-the-art climate-controlled greenhouse with an area of 5020 m2, which has been considered as the world first integrated aquaculture-horticulture system in a symbiotic manner with zero pesticide usage and effluent discharge. This facility consists basically of fish rearing tanks, settling or clarifier and biofilter tanks, water pump, hydoponic units (i.e., gullies) and sump tanks. Asian seabass or Barramundi (Lates calcarifer) are raised in multiple tanks below the hydroponic growing area. Effluent from fish tanks is biologically processed using a patented UESA bioconverter prior to irrigating the plants in the hydroponic unit in a continuous manner. In this experiment, the setup was arranged in randomized complete block design with three replications. Each growing gully was assigned as replicate consisting of 32 pots with at least 12 plants per pot. Growing gullies were programmed to move through an automated system throughout the growing cycle from transplanting of the seedlings until harvesting of marketable size. Correspondingly, irrigation was also
programmed for rate and frequency based on the plant sizes and requirements. Climatic parameters were set by the company based on the compromised requirements of two biological systems, plants, and fish. All other care and maintenance needed for both systems were done by their farm expert and technician following the company’s standard practices. Measurements and analyses Leaf number, plant height, stem diameter, leaf area, chlorophyll and protein contents, plant biomass, and total phosphorus and nitrogen were measured from both seedlings and plants from aquaponics. The levels of endogenous plant IAA, peroxidase and catalase activities were also measured. All these measurements were taken from at least 25 randomly selected plants per replicate at transplanting and at harvest from aquaponics with the exception of root length, which was only possible at seedling stage. Plant height was measured from the base of the plant to the top of longest leaf using a stainless ruler. Stem diameter was taken above the cotelydonary leaf and second leaf node at seedling stage and maturity (at harvest), respectively, using a digital Vernier caliper. Leaf area was measured using a portable leaf area meter (LI-3100C, LI-COR Inc., Lincoln, NE). Seedling roots were scanned using a Desk Scan II scanner (Expression 700, Epson, Nagano, Japan). Scanned root images were analyzed by WinRhizo Pro V. 2007c (Regent Instruments Inc., Quebec, Canada) for total root length measurement. After the measurements, roots and shoots were washed and oven-dried for 3 days at 70 °C for total N and P determination using CNS Vario max analyzer (Elementar Analysensysteme GmBH, Hanau, Germany) and colorimetric technique (ammoniun vanadateamonnium molybdate color reagent technique), respectively. Levels of endogenous plant IAA were measured following the method described by Ribaudo et al. (2006) with few modifications, while peroxidase activity was assayed colorimetrically following the method of BenShalom et al. (2003) with few modifications. Details of the modified procedures can be found in the report of Mangmang et al. (2015). Root colonization was assessed by collecting root samples, including those substrates loosely adhering to the roots, from randomly selected inoculated plants in each treatment. Collected samples were homogenized with 1 ml peptone phosphate buffer using a tissue lyser. An aliquot of the suspension was
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plated after a series of dilution on a NFB agar medium with congo red (Bashan et al. 1993). After 4 days of incubation at 28 °C, the number of cfu with distinctive color morphology of the test strain were counted and expressed per gram of root fresh weight (Gamalero et al. 2008). Data were analyzed following analysis of variance (ANOVA) using Genstat® 14th edition software (VSN International, Hemel Hempstead, UK). Mean differences were determined using least significant difference (LSD) (P
