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- Materials and methods
In Tunisia, great brome (Bromus diandrus Roth., syn.Bromus rigidus Roth. subsp. gussonii Parl.) is widely distributed in cereal crops resulting in yield losses that can reach up to 80% in heavily infested wheat-growing areas (Souissi et al. 2000, 2001). Control methods commonly used to suppress brome growth in wheat crops are essentially chemical and cultural. However, these methods are prohibitively expensive, and there are no selective herbicides for the control of brome in wheat (Mazzola et al. 1995; Souissi et al. 2000). In addition, excessive use of chemical herbicides has resulted in the development of herbicide resistance in many weed biotypes (Heap 2012).
In order to overcome these limitations, efforts have been made to develop alternative and more effective measures to manage weeds. Biological control of weeds with living micro-organisms was reported to be a good alternative to chemical treatment. Deleterious rhizobacteria (DRB) are among the micro-organisms that have been reported to have a potential as biocontrol agents in controlling weeds (Elliott and Lynch 1985; Kremer 1987; Schippers et al. 1987; Kremer et al. 1990; Kennedy et al. 1991; Kremer and Kennedy 1996; Kremer 2000; Kennedy et al. 2001; Flores-Vargas and O'Hara 2006; Li and Kremer 2006; Kennedy and Stubbs 2007; Banowetz et al. 2008; Mejri et al. 2010). However, successful application of live bacteria in a field setting depends upon many factors including environmental conditions and soil survival. Therefore, there is a need to formulate these biocontrol agents to enhance their field potential and facilitate their storage and application. Many weed biological control agents have been formulated into liquid, solid and powder substrates (Green et al. 1998) because of the low bacterial survival in liquid inoculants (Singleton et al. 2002; Tittabutr et al. 2007; Albareda et al. 2008) and impractical use of cell suspensions for large-scale application due to the difficult handling, transport and storage of the inoculum (Rabindran and Vidhyasekaran 1996; Vidhyasekaran et al. 1997a). A formulated bioherbicide can be defined as a mixture of the active ingredient (the biological agent) within a carrier or solvent that delivers the active ingredient to the target weed, and the adjuvants that improve the survival and effectiveness of the product in adverse environmental conditions (Boyette et al. 1991; Hynes and Boyetchko 2006; Chutia et al. 2007; Ash 2010). Among the different possible formulations, the dry solid or powder formulations provide several advantages. Bacterial cells immobilized in dry carrier are protected from the external environmental factors and their survival and efficacy are preserved in adverse environmental conditions (Boyette et al. 1991; Shabana et al. 2003; Kinay and Yildiz 2008). Dry carriers also allow efficient and easy delivering of bacteria to the target weed (Sabaratnam and Traiquair 2002). For instance, the wheat-gluten matrix known as Pesta has been used to formulate granular biocontrol agents. This matrix is adaptable to many different micro-organisms and ingredients (Daigle et al. 1997) and is nontoxic, cost-effective and easy to store and use (Elzein et al. 2004). Pesta formulations have been widely used to deliver mycoherbicides such as Colletotrichum truncatum against hemp sesbania (Sesbania exaltata) (Connick et al. 1991, 1996) and Fusarium oxysporum against sunflower broomrape (Orobanche cumana) (Shabana et al. 2003) and Striga spp. (Elzein et al. 2004). Pesta formulations for bacteria with bioherbicide activities have also been used with Pseudomonas fluorescens BRG100 against green foxtail (Setaria viridis) (Daigle et al. 2002), P. fluorescens strain G2-11 against velvetleaf (Abutilon theophrasti) (Zdor et al. 2005) and P. fluorescens LS102 and LS174 against leafy spurge (Euphorbia esula) (Brinkman et al. 1999). Talc powder dry formulations have been considered as a simple and cost- and time-effective technique. It has been mainly used to formulate bacteria (Hofte et al. 1991; Vidhyasekaran et al. 1997a) for large-scale field applications in different crops for the management of soilborne plant pathogens (Rabindran and Vidhyasekaran 1996; Vidhyasekaran et al. 1997a,b).
We have previously described the isolation, identification and physiological characterization of the deleterious rhizobacterium, Pseudomonas trivialis X33d, a promising biocontrol agent against great brome in wheat (Mejri et al. 2010). The main objectives of this work were to develop an appropriate formulation of the strain X33d and to assess its efficacy in inhibiting great brome growth under controlled and greenhouse conditions. In addition, the storage conditions (i.e. temperature and addition of adjuvants) required in order to improve the survival of X33d in the formulation were standardized.
- Top of page
- Materials and methods
Weed biological control by specific micro-organisms is an accepted strategy for weed management (El-Sayed 2005; Caressa et al. 2010). The deleterious rhizobacteria (DRB) belonging to the Pseudomonas group are known to be promising candidates for weed biological control (Kennedy et al. 1991; Kremer and Kennedy 1996; Flores-Vargas and O'Hara 2006). Cell suspension of these DRB has been found to be an effective bioherbicide. However, the use of bacterial cell suspensions in large field scale is hard to manage due to difficulty in handling and storage. Therefore, development of robust formulations, ensuring simple handling, long shelf life and high cost-efficiency, can help to overcome such difficulties. The selection of the appropriate carrier, enabling the dispersion of the biological material to the target plant, is essential for the successful development of a formulation (Nakkeran et al. 2005).
Pseudomonas trivialis X33d is able to reduce the growth (in the terms of dry weight) of the weed Bromus diandrus while increasing the development of durum wheat. Physiological traits possibly involved in this dual behaviour have been discussed in the study by Mejri et al. (2010). In this work, the impact of cell suspension of Ps. trivialis X33d on great brome growth was assessed and compared with the effect induced by ‘Pesta’ granules and talc powder formulations of the bacterial strain. Our results showed that both the bacterial formulations and the cell suspension reduced great brome growth compared to uninoculated controls. The bioherbicide activity of cell suspension and ‘Pesta’ granular formulation of X33d was similar, which may suggest that the phytotoxic activity of X33d was not affected during the formulation process. In addition, our results showed that the cell viability of X33d was higher in ‘Pesta’ granules than that in talc powder formulation. This result shows that cell viability of the biological agent may be affected by the type of the carrier in the formulation.
The active ingredient of a bioherbicide is sensitive to many variables throughout formulation (Connick et al. 1996). Among them, temperature is one of the main factors contributing to the quality of the formulation, which is reflected in the efficacy and the long shelf life of the bioherbicide. Hence, the shelf life of the strain X33d formulated as ‘Pesta’ granules was evaluated during storage at two different temperatures (room temperature and 4°C). According to the literature, ‘Pesta’ granules were considered to be nonviable if the density of bacterial cells in the granules is less than 1 × 105 CFU g−1 (Daigle et al. 2002). Our results demonstrated that the concentration of bacterial cells in ‘Pesta’ granular formulated Ps. trivialis X33d stored for 6 months was higher than 105 CFU g−1, irrespective of the storage temperature. Moreover, bacterial viability in ‘Pesta’ granules stored at 4°C was higher than that measured in granules stored at room temperature. In fact, both cell division and metabolic rate in bacteria are slowed down by storage at low temperatures (4–10°C). In this condition, the depletion of nutrients is reduced and the accumulation of toxic metabolites and the loss of moisture in the carrier are prevented, therefore favouring the long-term storage of the bacterial inoculants (Van Shrevan 1970; Kirsop and Doyle 1991; Trivedi et al. 2005). Consequently, low temperatures of storage have been successfully used for many formulated fungal and bacterial biological control agents (Kirsop and Doyle 1991). For instance, excellent recovery of Colletotrichum truncatem, Alternaria cassiae and A. crassa in ‘Pesta’ granules was observed after 18 months of storage at 4°C (Connick et al. 1991). The survival of Colletotrichum dematium FGCC# 20 in ‘Pesta’ granules after 1 year was found to be higher at 4°C than at room temperature (Singh and Pandey 2010).
The viability of living organisms in granular formulations during storage may be affected by the nutritional amendments added to the formulation (Shabana et al. 2003). It has been suggested that the Pesta production process is amenable to incorporation of many solid and liquid additives, which may reduce the cost and alter certain properties of the final formulation (Shabana et al. 2003). The survival of Ps. trivialis X33d formulated in ‘Pesta’ granules amended with sucrose (+S-O-) or oil (-S+O) fully overlapped and were higher than in granules without adjuvant (-S-O). Moreover, the addition of both sucrose and oil (+S+O) to ‘Pesta’ granules induced a synergistic effect on the density of the strain X33d in ‘Pesta’ granules compared to all the other adjuvants, probably due to the provision of additional nutrients. Sucrose is known to extend bacterial survival throughout storage because it has a putative role as membrane stabilizer during the drying (Caesar and Burr 1991; Leslie et al. 1995; Connick et al. 1996), and oil has been shown to enhance the survival of fungi and nematodes in alginate formulations (Quimby et al. 1994; Caesar-Tonthat et al. 1995). Our results partially corroborate the finding of Zidack and Quimby (2002) who reported that the long-term survival of Pseudomonas syringae pv. tabaci and Pseudomonas syringae pv. tagetis in Stabileze formula differs according to the adjuvant added. While the long-term survival of P.s. pv. tabaci was enhanced by oil alone and sucrose and oil in combination, survival of P.s. pv. tagetis was not enhanced by oil, and oil in the formulation reduced the beneficial effects of sucrose. Moreover, the addition of sucrose to Pesta granules partially counteracted the detrimental effect of high water activity on the shelf life of Colletotrichum truncatum (Connick et al. 1996). Finally, granular ‘Pesta’ of Fusarium oxysporum sp. orthoceras supplemented with yeast extract and sucrose showed improved mycoherbicidal efficacy, stability and shelf life over time (Shabana et al. 2003).
Taking into account the results obtained, we decided to assess, under controlled conditions, the efficacy of Pesta granules of Ps. trivialis X33d with or without the two adjuvants and stored for 6 months at 4°C in suppressing great brome growth, without showing any negative effect on durum wheat. The results obtained showed that brome development was reduced by ‘Pesta’ granules of the strain X33d, this inhibition being stronger in ‘Pesta’ granules supplemented with sucrose and oil. On the contrary, wheat biomass was increased in plants inoculated with the ‘Pesta’ granules of the bacterial strain, this promotion being higher in the presence of both adjuvants. The stronger effects on plant growth (suppression of brome and stimulation of wheat) recorded in plants treated with ‘Pesta’ granules of Ps. trivialis X33d added with sucrose and oil are consistent with the observed high survival, and rhizosphere competence, of the bacterial strain in granules supplemented with both the adjuvant and stored at 4°C.
Because the bioherbicide effects and plant growth promotion could be affected by environmental conditions, a further experiment was performed by growing brome and wheat plants in the greenhouse inoculated or not with ‘Pesta’ granules of X33d with adjutants. The results obtained showed that the bioherbicide activity of this bacterial formulation on brome growth and yield and the plant growth–promoting effect on wheat growth and yield were maintained also in uncontrolled environment conditions. Therefore, the efficacy of the ‘Pesta’ granular formulation with adjuvants of the strain X33d was unaffected by the 6-month storage at 4°C. However, the reduction of great brome biomass, as well as the stimulation of wheat biomass, was more evident under controlled conditions than in natural ones. This is consistent with the impact of environmental conditions on the survival, efficiency and rhizosphere competence of micro-organisms behaving as DRB or plant growth–promoting bacteria (PGPB) (Kremer 2005).
This work has demonstrated the successful use of the deleterious rhizobacterium Ps. trivialis X33d formulated in ‘Pesta’ granules as a bioherbicide against great brome. The use and application of such bioformulations in the field can result in the reduction of application of harmful chemicals, protect the environment and biological resources and be an important component of integrated pest management in sustainable agriculture. This investigation is only the first step in order to obtain a suitable formulation for application in the field after storage. Other research needs to be addressed before large-scale applications. In particular, further work will be performed to (i) assess the survival and the efficacy of ‘Pesta’ formulation of X33d in soils with different chemical and physical characteristics and (ii) analyse the root colonization pattern of the strain X33d formulated in ‘Pesta’ granules or as free cells, in brome and wheat.