IJERD – International Journal of Environmental and Rural Development (2012) 3-2

Research article
erd

New Biotechnologies for Sustaining Greener Agriculture
IVAN R. KENNEDY*
Faculty of Agriculture and Environment, University of Sydney, Australia
Email: ivan.kennedy@sydney.edu.au

NGUYEN THANH HIEN
Biofertilizer Action Center, Hanoi, Vietnam

PHAN THI CONG
Agriculture Green Future, Ho Chi Minh City, Vietnam

KHANOK-ON AMPRAYN
Faculty of Agriculture and Environment, University of Sydney, Australia

GANISAN KRISHNEN
Faculty of Agriculture and Environment, University of Sydney, Australia

MICHAEL ROSE
Faculty of Agriculture and Environment, University of Sydney, Australia

ANGUS CROSSAN
Faculty of Agriculture and Environment, University of Sydney, Australia

ZHANG YAN

Tianjin University of Science and Technology, Tianjin,  People’s  Republic  of  China  WANG SHUO
Tianjin University of Science and Technology, Tianjin, People’s  Republic  of  China
Received 13 February 2012

Accepted 12 March 2012(*: Corresponding Author)

Abstract Sustainable agriculture must face the challenge of sufficient productivity to feed
and clothe up to 10 billion people this century. As a result, strictly organic methods, whileenvironmentally welcome, can be no more than a partial solution because more land isneeded than is available. However, recent development in a range of biotechnologies may
provide safer environmental protection together with improving yields. For example, plant
growth-promoting (PGP) microorganisms in Vietnam have been shown in field studies tooffer reduced needs for seed, chemical fertilisers, pesticides, water and harvest yield losses.
Interestingly, one PGP inoculant called BioGro was designed using validation of microbial
strains from farmers' results; but these very same species are now being characterised inlaboratories world-wide as PGP strains. Their laboratory analysis has shown similar traits
as observed by farmers, such as extended root structure, nutrient mobilisation, biocontrol
for pathogens and insecticidal activity. Despite less nutrient and pesticide needs and thus
reduced environmental impacts, agrochemicals will still be needed for sufficient productivity. Research on tools for environmental risk management by farmers such as immunological ELISA analysis can also contribute here. Rapid 5-minute tests for monitoring bothfood and environment for pesticides can provide better stewardship of such chemicals,
helping  to  ensure  a  ‘greener  future’.
Keywords nitrogen fixation, PGPR, safer pesticides, immunogold tests, risk managementⒸISERD88

IJERD – International Journal of Environmental and Rural Development (2012) 3-2INTRODUCTION
In this paper, two recent areas of biotechnology developments now being applied will be described.
These are based on original research findings made in our group at the University of Sydney, inVietnam and in China since around 2000 or earlier, leading to the application of these findings andtheir launching in a commercial format. More than 10 years has been required for this result, given
the need for robust field research and extensive quality control of all outputs.
The two areas that will be described cover plant growth promoting microbes used to achieve
nutrient efficient rice production and the development of rapid test kits for measuring pesticides
used in crop production. Both of these outcomes are directed towards achieving more sustainableagriculture, by reducing environmental impacts and improving food safety.
MICROBIAL BIOFILMS AND PGPR RESOINSES ON RICE YIELDSMicrobial biofilms play an important role in agriculture and in food and fibre production. Whilst
some microbial effects on crop plants are deleterious, it is increasingly recognised that biofilms
may have strong beneficial effects in agriculture. Furthermore, realising these potential benefits islikely to play an increasing role in achieving sustainable solutions regarding improved crop yields
and in human and environmental health. Such gains may be essential in the face of the increasing
global population and climate change.
Plant growth promotion (PGP) for crops by microorganisms has been recognised for sometime in biological nitrogen fixation (BNF) in legumes, shown in Germany in the late 19th century in
the face of opposition by Justus Von Liebig. More subtle are the beneficial effects of the PGP microbes in cereals and other crops. These involve a complex of phytohormonal effects, nutrient mobilisation, BNF and biocontrol that benefit plant growth and overall yield (Kennedy et al. 2008).
Essentially, it is possible to grow crops such as rice inoculated with microbes forming biofilms onthe surfaces of their roots that allow significantly reduced input of chemical fertilisers, seed, pesticides and water, as well as reduced harvest losses.
In this paper we describe a successful biofertiliser, BioGro - now a commercial product in Vietnam invented by Professor Nguyen Than Hien; it consists of four microbial strains in peat (Pseudomonas fluorescens, Bacillus spp. and a soil yeast, Candida tropicalis) inoculated to the rhizosphere of rice seedlings (Nguyen et al., 2003; Phan et al., 2009, 2011). Remarkably, BioGro contained unidentified strains selected empirically on the basis of their effectiveness for rice farmers;
later, these were identified in Australia to include species now recognised around the world in research laboratories as having a strong genetic basis for beneficial effects as inoculant biofertilisers(Kennedy et al., 2008).
As was the case about a century ago with legumes and the rhizobia that form N2-fixing nodules on their roots, obtaining an effective outcome from applying these biofilm-forming organisms
to crops in the field is challenging. It is important that inoculants of high quality in terms of particular microbial strains in sufficient numbers (ca. 107 -108 cells per g of inert carrier). Furthermore,convincing farmers of the beneficial effects and the scale of economic benefits possible, limiting
their risk, is also important. An efficient supply chain that includes quality control and delivery ofeconomic and environmental benefits from biofertilisers to farmers is also essential.
A strategy for simultaneously achieving environment-friendly and economic benefits developed in a World Bank Development Marketplace project Sustaining nitrogen-efficient rice production (DM2008), was developed. Recent unpublished research indicates the role of cyclic lipopeptides and phenazines produced by Pseudomonas sp. CMR12a in root colonization, biofilm formation and biological control of soil borne plant pathogens, regulation of insect plant-beneficial
pseudomonad and insecticidal activity of plant-associated pseudomonads: how they became insect
pathogens, aiding plant salt tolerance by tissue specific regulation of sodium uptake, thus conferring plant growth promotion with yield increases from less fertiliser (Nguyen et al., 2003; Phan etal., 2009, 2011) in the application of such microbes to crops as biofertilisers like BioGro still needs
to be determined.
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IJERD – International Journal of Environmental and Rural Development (2012) 3-2

However, the results of a participatory action process with the application of BioGro at village
level in the Mekong Delta has led to significant increase in the profitability of rice farming (Fig. 1).
Fig. 1 shows plots of yield ratio to profit ratio that farmers using BioGro replacing about 40% ofurea application can obtain as greater yields and profits as a result of better tillering, lower Nfertiliser inputs, reduced pesticide inputs, improved water use efficiency and reduced harvest losses
as machine harvesting could occur more promptly. The tendency to be in the top sector with ratios
greater than 1.0 increased with experience, most of the values falling below 1.0 occurring in thesummer-autumn crop of 2009 during the third crop before the annual flood in September when soil
condition was acid sulphate and nutrients were low and soil toxicity greatest. Crops on the sameplots twelve months later nearly all had ratios exceeding 1.0.
2.2

Profit ratio (BioGro/Farmer)

2.0
1.8

R2 = 0.8
P< 0.0001
Y = -1.36 + 2.4X

1.6
1.4
1.2
1.0
0.8
0.6
0.4

0.98

0.8

1.0

1.2

1.4

Yield ratio (BioGro/Farmer)

Fig. 1 Yield and profit ratios over four crops comparing farmers using 100 kg of BioGro
reducing urea inputs to about 60% compared to normal farmer practice (100 kg
urea-N per ha) on individual plots less than 1 ha are shown. TAn imperative here for success of this biotechnology shown in Fig. 1 with BioGro is ensuring
the quality control of these inoculants; the correct strain species in the correct numbers must bepresent in commercial inoculant products. Using techniques similar to those employed to produce
immunogold tests for herbicides described in more detail below, we have also generated similar
immogold tests for beneficial PGPR microbes such as Pseudomonas fluorescens, one of the key
components of BioGro (see Fig. 1).

Fig. 2 Rapid immunogold quicktests for 1N (Pseudomonas fluorescens), a component ofBioGro used in rice production
The  test  lines  (T)  show  increasing  concentrations  of  1N  (cfu’s  per  mL).  These  devices  are  being applied to testing commercial peat cultures as part of their quality control, ensuring that specificstrain cell numbers exceed 106-107 viable cells per g.
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IJERD – International Journal of Environmental and Rural Development (2012) 3-2

QUICK IMMUNOTESTS FOR HERBICIDES
In our research, three polyclonal antibodies against herbicides used in cotton growing, diuron,
fluometuron and prometryn for the preparation of rapid lateral flow immunogold tests have recenly
been developed. We will provide some details of the preparation of two of these rapid tests, for diuron and prometryn.
1. DiuronA rapid immunotest for diuron was developed using published techniques. Haptens were synthesised as indicated. The hapten was attached to the protein carriers of keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA), ovalbumin(OVA) and horseradish peroxidase (HRP) using
the active ester method. The conjugates of hapten 4C-KLH, hapten 6C-KLH, hapten 4C-BSA andhapten 6C-BSA were all used as immunogens.

Fig. 3 Preparation of haptens for antibodies reactive with diuron
Sensitivity of the test could be controlled by varying the length of the coupling groupused for conjugation to protein for injection into rabbits

A pair of male rabbits was immunized with each immunogen. Test results showed that rabbits
immunized with the conjugate of hapten 4C-KLH produced the best specific antibody. To test thesuitability of the antibodies, competitive indirect ELISA microtiter plates were coated with 100 μL  per well of hapten 4C-OVA  (0.1  μg mL−1 in 50 mM carbonate buffer, pH 9.6) overnight at 4 o C.
The following day, the plates were washed with PBST and blocked with 200 μL  per  well of 0.5%
milk powder diluted in PBS.  50μL  per  well  of  diuron  and  50  μL  per  well of rabbit anti-diuron pAb(1  μg  mL−1, diluted in PBS) were added. Plates were shaken gently for one minute before incubatedfor 1 h at 37 o C.  Then  the  plates  were  washed  again  with  PBST,  and  100  μL  per  well  of  10000-folddiluted secondary antibody were added and incubated at 37 o C for 30min. After another washing
step,  150  μL  per  well  of  substrate  were  added  and  incubated  for  15  min.  The  enzyme reaction wasstopped with 1.5 M H2SO4 and the plates were read at 450 nm (reference 650 nm). Having shown
the suitability of the IgG, immnogold strip tests were prepared. Full details of their manufacture
will be published elsewhere.

Fig. 4 Diuron Quick Test
As test water samples contain diuron as a contaminant, the test line (T) disappears,The control line, containing non-specific antibody (C) indicates the antibody test is effective

2. Prometryn
The following is the chemical structure of the hapten for prometryn:ⒸISERD91

IJERD – International Journal of Environmental and Rural Development (2012) 3-2

The hapten synthesis is as the following:

The hapten was attached to the protein carriers bovine serum albumin (BSA), using the Nhydroxysuccinimide active ester reaction Hapten-BSA was used as immunogen. The titre of theserum produced was 160,000, which was purified by protein A-sepharose-4B.
The optimization of a direct competitive ELISA for prometryn is ongoing, under the preliminary condition of ELISA, IC50 is 0.4 ppb.

Fig. 5 The standard curve of direct competitive ELISA for prometryn
Immunogold tests for pesticides based on these ELISA results are currently in preparation

CONCLUSION
Farmers need many new tools to assist them to achieve sustainable agricultural practices. In thispaper, we have described two areas where biotechnology has allowed advances that both help
achieve higher crop yields for rice and also allow farmers to manage environmental risk regarding
their application of agrochemicals. We plan to generate a large range of such rapid tests, including
some for screening food products for aflatoxin produced by Aspergillus flavus. The availability ofsuch rapid tests at a reasonable cost will provide tools supporting best management practices.
Organic production of crops is a worthwhile aim when consumers of farm products can afford
to pay the higher costs. Organic production requires at least 50% more land than conventional agriculture so that only a small proportion of organic products can be expected without clearing moreforests. With global climate change, this is unacceptable. But feeding the world sustainably will
require most food to be grown using all farming tools available, leaving choices of the best production systems to farmers. Government agencies must support farmers to make the best choice and toundertake stewardship. Banning chemical products and farming practices such as land clearing iscounterproductive. Far better for farmers to volunteer protection of the environment, using modern
biotechnology such as that described in this article.
REFERENCESCong, P.T., Dung, T.D., Hien, T.M., Hien, N.T., Choudhury, Abu T.M.A., Kecskés, M.L. and Kennedy, I.R.
2009. Inoculant plant growth-promoting microorganisms enhance utilisation of urea-N and grain yield ofpaddy rice in southern Vietnam. European Journal of Soil Biology, 45, 52-61.
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Hien, N.T., Deaker, R., Kennedy, I.R. and Roughley, R.J. 2003. The positive yield response of field-grown
rice to inoculation with a multi-strain biofertiliser in the Hanoi area, Vietnam. Symbiosis 35, 231-245.
Kennedy, I.R., Choudhury, A.T.M.A., Kecskes, M.L. and Rose, M.T. (eds.) 2008. Efficient nutrient use inrice production in Vietnam achieved using inoculant biofertilisers. Proceedings of a project(SMCN/2002/073) workshop held in Hanoi Vietnam, 12-13 October 2007, ACIAR Proceedings, No. 130
137. Canberra ACT. www.aciar.gov.au.

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