Interactions between the plants and microorganisms

Allelopathy Journal 31 (1): 51-70 (2013) Tables: 7, Fig : 1

0971-4693/94 US $ 5.00 International Allelopathy Foundation 2013

Interactions between the plants and microorganisms ROBERT J. KREMER USDA, Agricultural Research Service, Cropping Systems & Water Quality Unit, University of Missouri, 302 Anheuser-Busch Natural Resources Building, Columbia, MO, 65211 USA. E. Mail: [email protected] (Received in revised form: November 30, 2012)

CONTENTS 1. INTRODUCTION 2. COLLECTION OF ALLELOPATHIC MICROORGANISMS 2.1. RHIZOBACTERIA 2.2. RHIZOSPHERE FUNGI 3. ASSESSING ACTIVITY OF ALLELOPATHIC MICROORGANISMS 3.1. SEEDLING BIOASSAYS FOR RHIZOBACTERIA 3.2. SEEDLING BIOASSAYS FOR RHIZOSPHERE FUNGI 4. CONCLUSIONS 5. REFERENCES

ABSTRACT Allelopathic microorganisms comprise rhizobacteria and fungi that colonize the surfaces of plant roots and produce and release phytotoxic metabolites, similar to allelochemicals, that adversely affect the growth of their host plants. The allelopathic microorganisms are grouped separately from typical phytopathogens because they do not follow classical disease cycles involving aggressive infection and colonization of plant tissues. Allelopathic microorganisms passively suppress or inhibit the plant root growth through production of variety of phytotoxic compounds absorbed by the plant, or they compete for limited plant nutrients in the rhizosphere. Thus it is important to understand the effects of allelopathic microorganisms on plant growth and discern these from traditional phytopathogens so that appropriate management of plant growth problems can be implemented based on this knowledge. The purpose of this paper is to provide basic information on protocol development of bioassays to identify allelopathic microorganisms associated with plants. Guidance provided by the general procedures is presented as templates for devising custom protocols to aid in eventual development of standardized bioassays for consistent detection and characterization of allelopathic microorganisms, for which little information and reliable data currently exist. The use of improved bioassays that yield reproducible results will enhance comprehension of the effects of allelopathic microorganisms on plant growth and increase the overall perspective of functional biology and ecology of the rhizosphere microbial community beyond general descriptions of interactions between plants and soilborne phytopathogens. Key Words:

Allelopathic microorganisms, bacteria, fungi, Fusarium spp., hydroponics bioassays, rhizobacteria, rhizosphere, seedling bioassays, soilborne phytopathogens

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1. INTRODUCTION A variety of environmental microorganisms resides in soil on plant seeds and roots of seedlings. Most soils are deficient in nutrients but the soil zones surrounding the seed (spermosphere) and root (rhizosphere) provide micro-environments with organic substrates to readily make available nutrients for soil microorganisms (11). Hence, the spermosphere and rhizosphere are ideal sites, to isolate the selected microorganisms (suppress seed germination and seedling growth) due to the continuous supply of carbon and energy released from germinating seeds and developing roots (34). Soilborne bacteria adapted to colonization of spermosphere, rhizosphere and the root are called rhizobacteria (27). The spermosphere (seed zone before root emergence), contains the substances exuded from the seed, establishes the dominant microbial communities of the rhizosphere (22). Some deleterious rhizobacteria and rhizosphere fungi in spermosphere and rhizosphere inhibit the plant growth, without causing disease symptoms. These deleterious rhizosphere microorganisms (DRMO) are mainly saprophytes [Survive on organic compounds released from sown seeds and root cells (26)], hence, live on plant seeds and roots. DRMO neither parasitizes the plant nor penetrates the stele like major pathogens, but colonize the seed tissues or root hairs, root tip and the intercellular spaces of root cortical cells (26)] and are called “Endorrhizal DRMO.” Many DRMO release phytotoxic metabolites or allelochemicals, that influence the plant growth, thus called “allelopathic microorganisms” (4,13). DRMO that reduces the seed germination and seedling development of plants through production of allelochemical substances may be called allelopathic bacteria and allelopathic fungi, respectively. This article describes the selected protocols to characterise and evaluate the effects of allelopathic microorganisms on plant growth (15,19). Selection of allelopathic microorganisms is based on standard microbiological and phytopathological methods and the effects of selected microorganism are assayed using either a specific or a model plant specie as an indicator of allelopathic activity. Assays are done as preliminary screenings in plate culture to quickly assess the effects; isolates exhibiting the positive allelopathic effects are then subjected to secondary screening, which involves use of hydroponic and culture filtrate systems.

2. COLLECTION OF ALLELOPATHIC MICROORGANISMS Allelopathy may be exploited for biological weed management in various agricultural and natural ecosystems. A component of such biological weed management systems is the use of living microorganisms including bacteria and fungi to reduce the vigour, reproductive capacity, density, or overall impact of weeds (13). Thus, protocols have been developed to detect and characterize microbial candidates for allelopathic effects as potential biological control agents. These protocols are presented in the following section.

Plant-microorganisms Interactions

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2.1 RHIZOBACTERIA Rhizobacteria communities are rich sources of allelopathic bacteria due to their intimate association with plant roots. The following protocols illustrate how this group of bacteria can be efficiently isolated and characterized for allelopathic activity. 2.1.1. Isolation and Characterization of Rhizobacteria: To determine if allelopathic effects are due to specific bacteria, individual species associated with the affected plant must be isolated and characterized prior to screening on target plants. The following is a general scheme for isolating rhizobacteria to provide pure cultures originating from test plants that can then be used in numerous subsequent screening trials (15). This scheme provides a basic outline that can be later modified to meet the investigator’s objectives by accommodating different plant structures (i.e., seeds, roots, rhizomes, culms, etc.), different plant growth stages, or specific bacterial groups using other and/or multiple selective culture media. Materials and equipment required Freshly-collected seedling roots, trowel, soil probe (optional), plastic sample bags, cooler, seeds, soils, soil sieve, test tubes, tube mixer (Vortex Genie), analytical balance, autoclave, pipettes, petri plates, glass rod spreaders, bacteriological loops, marking pens, Bunsen gas burner or alcohol burner, incubator (optional), pots (7.5-cm diam) for greenhouse (optional) Reagents and culture media required 0.01M MgSO4 buffer: Dissolve 1.203 g of reagent-grade MgSO4 in 1L of distilled water and store in reagent bottle under ambient conditions; 95% ethanol; S1 agar medium (see Table 1) Table 1. Composition of S1 culture medium for rhizobacteria isolation and maintenance (9) Ingredients Quantity Agar 18.0g Sucrose 10.0 g Glycerol 10.0 mL Casamino Acids 5.0 g NaHCO3 1.0 g MgSO4 . 7H2O 1.0 g K2HPO4 2.3 g Sodium lauryl sarcosine (SLS)** 1.2 g Trimethoprim*** 20.0 mg De-ionized Water 1000 mL Medium contents has final pH of 7.4 - 7.6, not necessary to adjust pH. **SLS is stored in desiccator jar , ***Prepare stock of trimethoprim and add by filter-sterilization to medium after autoclaving.

Procedure (i). Collect intact plant seedlings from field sites by carefully removing with trowel, placing seedling with soil attached to roots in a plastic sample bag (Ziplock sandwich-

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or quart-size), and placing bags into insulated cooler for transport to laboratory. If field air temperatures >30 °C, a ‘cold pack’ or ice should be included in the cooler to prevent heat damage to the seedlings. Optional method: Collect soils from the selected field sites using soil probe or trowel to sample from upper 10 cm soil profile; place soil into plastic sample bag, place in cooler for transport to lab. Pass soil through coarse sieve (i.e., 2-mm mesh), dispense into 7.5-cm diam pots, plant seeds of target plant in soil, place in greenhouse allow seedlings to develop. After 14 days or at seedlings’ first fully developed true leaf, carefully remove and handle as outlined above. (ii).

Carefully remove seedlings from sample bag, gently shake to remove loosely held soil, cut top growth at ‘soil line’ using flame-sterilized scalpel or scissors and place roots in test tube containing 10 mL of sterile 0.01M MgSO4 buffer. (iii). Rigorously shake tube containing plant root using a vortex mixer (set at a high speed) for one minute. Immediately after mixing, transfer 1.0 mL of root soil suspension to 9.0 mL of buffer in test tube; continue transferring 1.0 mL aliquots from successive tubes to make a 10-fold dilution series up to a 1:1,000,000 or 10-6 dilution endpoint (24). Mix each tube prior to transfer and also use a new pipette for each transfer. (iv). Set up previously prepared S1 culture medium plates for transfer of diluted aliquots by labeling plates with seedling code name and dilution; use at least duplicate plates for each dilution. Pipette 0.1 mL (100 µL) from each of selected dilutions to be plated to the centre of the agar plate (i.e., final three dilutions, 10-4, 10-5, and 10-6 should yield plates with well-isolated single bacterial colonies, however, it may require preliminary plating of wider range of dilutions to determine the minimum number to plate). (v). Immediately spread the aliquot using a flame-sterilized spreader (“L-shaped” glass rod) – immerse bent portion of spreader in 95% ethanol, flame with burner, allow to cool. If aliquot is not spread quickly, it will absorb into the agar resulting in nondifferentiated bacterial growth in middle of plate. (vi). Repeat plating for each dilution series for all test seedling root suspensions. Sterilize spreaders between plates and different dilutions. (vii). When plates have dried, invert and place in incubator set at 24 to 26C or if ambient conditions are within this range, place on a clean laboratory bench remote from routine use. (viii). After 48 -72 h, examine plates for bacterial colony development and lack of contamination. For each seedling, count replicate plates at one dilution that contains between 30 and 300 colonies. Record colony counts for calculating rhizosphere bacterial numbers (populations). (ix). From any of the plates with colonies well separated from neighboring colonies, select colonies of interest by touching a flame-sterilized bacteriological loop to the colony to remove a small amount of culture. (x). Open a fresh S1 culture plate and quickly make a streak of the transferred culture by dragging the loop across the agar surface in one direction, re-sterilize the loop, cool, touch loop at end of first streak, drag the loop in a streak at 90° to original streak,


(xi).

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re-sterilize the loop, cool, touch loop at end of second streak, drag the loop in a streak at 90° to second streak. Incubate plates; after 48 h, individual (pure) colonies should grow in final streak and can be isolated in pure culture onto a fresh S1 culture plate. Isolates collected in this manner can be identified and assayed for allelopathic activity. Pure cultures can be characterized based on standard procedures (8,29) to include Gram stain, oxidase reaction motility and morphology. Taxonomic identification can be done using standard physiological assays (16) or molecular determinations (2).

Observations Although S1 medium is formulated for selective culture of fluorescent pseudomonad bacteria, several other genera of gram-negative and –positive bacteria can be isolated (15). A diverse collection of presumptive allelopathic bacteria can be obtained using this procedure with diligent selection of a variety of colony types based on pigmentation, gross morphological features (size, elevation, and margin features of colonies; polysaccharide production [‘gummy or sticky’ nature], colony opaqueness or transparency). If fluorescent pseudomonads are preferred for isolation, exposure of plate to ultra-violet light will cause diffusible, water-soluble, yellow-green pigments produced by the colonies to fluoresce, allowing easy selection and isolation. Calculations Rhizosphere bacterial numbers may be expressed on root fresh weight or length basis or on soil weight basis. For example, if number of colonies resulting from three replicate plates at a 10-5 dilution is 30, 38, and 32: (30 + 38 + 32) / 3 = 33.3 colonies These grow at a 10-5 dilution and assume root length was 2.5 cm: Number of colonies per cm root = 33.3 / (10-5) (2.5 cm) = 1.33 X 10-6 or 1,330,000 2.2 RHIZOSPHERE FUNGI Many fungi associate with plant roots as saprophytes often involved in nutrient transformations that provide available nutrients to plants. Some of these fungi, however, may selectively injure certain plants and are candidates as biological weed control agents based on their allelopathic activity. The following protocols are useful in obtaining the potential fungal agents for subsequent screening in field studies. 2.2.1. Isolation and Characterization of Rhizosphere Fungi: Soilborne and plantassociated fungi constitute a very broad and diverse group of eukaryotic microorganisms, some of them may exhibit allelopathy, distinct from various pathogenic mechanisms leading to plant disease and mediated by known phytopathogenic fungi. The fungi Kingdom have > 70,000 fungal species, but we will focus on members in the phylum Ascomycota, which are mostly soilborne and/or associated with plants. In this phylum Fusarium species possesses allelopathic activities and have been extensively studied. The method to isolate Fusarium from plants and subsequent characterization is provided, to

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serve as basic scheme that can be modified for other fungal groups depending on the needs of research project. Materials and equipment required Freshly-collected roots from plants of desired growth stage, trowel, soil probe (optional), plastic sample bags, cooler, seeds, soils, clippers or pruning shears, artist’s brush, soil sieve, 250- and 600-mL beakers, sterile paper towels, test tubes, mixer, analytical balance, magnetic stirrer, autoclave, pipettes, petri plates, bacteriological needles, forceps, scissors or scalpel; marking pens, calipers or rulers, Bunsen gas burner or alcohol burner, microscopes (dissecting and light), incubator (optional), pots (7.5-cm diam) for greenhouse (optional) Reagents and culture media required 0.01M MgSO4 buffer: Dissolve 1.203 g of reagent-grade MgSO4 in 1L of distilled water and store in reagent bottle under ambient conditions; 1.25% Sodium hypochlorite (or 10% commercial bleach); 95% ethanol; sterile de-ionized water; Fusarium-selective agar medium (see Table 2); potato dextrose agar (PDA) medium (see Table 3) Table 2. Composition and preparation of Fusarium-selective culture medium (21) Ingredients Agar D-galactose L-asparagine MgSO4 . 7H2O KCl K2HPO4 Fe(EDTA) Distilled Water Inhibitors & Antibiotic Ingredients: Oxgall (“Oxbile” - Sigma # 70168) Pentachloronitrobenzene (PCNB) (“Quintozene” - Sigma #P220-5) Sodium tetra-borate (Na2B4O7*10 H20) (Sigma #S9640) Streptomycin sulfate (Sigma #S6501)

Quantity 15 g 20 g 2.0 g 0.5 g 0.5 g 1.0 g 0.005 g 1000 mL 0.5 1.0 1.0 0.03

Preparation and Precautions: 1. Dissolve all major ingredients except agar in distilled water and mix on magnetic stirrer. Adjust pH to 3.8 - 4.0 using phosphoric acid. Then add agar and autoclave (15 min at 121oC and 20 psi). 2. After autoclaving, allow medium to cool to 45 - 50oC; add the Inhibitor & Antibiotic Ingredients. 3. These ingredients are very toxic, therefore, use protective laboratory gear; it is not necessary to filtersterilize into the autoclaved medium. Suggest using sterile water, however, in preparing these ingredients for amending the medium. 4. PCNB is water insoluble – carefully dissolve in a minimal amount (20-25 mL) of benzene before adding (this should be done under a fume hood). 5. Oxgall and Na tetra-borate are very difficult to dissolve in water –suspend these ingredients together in sterile water (about 100 mL) and using magnetic stirrer, stir the mixture one hour prior to adding to autoclaved medium. 6. Streptomycin will readily dissolve with slow addition to the medium (this can be dissolved together with oxgall and sodium tetra-borate and added to medium together.

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7. 8. 9.

After all amendments made, stir molten agar to assure uniform dispersal but try to avoid foaming. Pour plates at 25- 30 ml per plate - need rather thick agar since incubations sometime last 1-2 wk and plates begin to dry. Despite all precautions with the amendments, after agar in plates set up (gel), white flakes or precipitates and occasional “oily spots” will appear in the agar. This is normal due to nature of ingredients and does not appear to affect the growth of fungi.

Table 3. Composition and preparation of potato dextrose agar (PDA) culture medium (22) Ingredients Potatoes (baking grade, white, peeled) Dextrose Agar Deionized water

Quantity 250 g 20 g 20 g 1000 mL

Preparation: Slice potatoes (avoid using red-skinned potatoes) into 500 mL water; add agar to 500 mL water in separate flask. Place both containers in autoclave for 45 min. Strain potato broth through several layers of cheesecloth into flask containing melted agar. Squeeze remaining potato pulp through several layers of cheesecloth until ca. 140 g potato pulp is obtained; add the pulp to the melted agar + potato water and add dextrose. Adjust total volume to 1000 mL by adding water. Mix all ingredients thoroughly, autoclave for 30 min. Remove from autoclave, allow contents to cool to ca. 40C, dispense ca. 25 mL into petri plates.

Procedure (i). Collect intact plant roots from field sites by carefully removing with trowel, place roots with attached soil in a plastic sample bag (Ziplock sandwich- or quart-size) and place bags into insulated cooler for transport to laboratory. If field air temperatures >30 °C, a ‘cold pack’ or ice should be included in the cooler to prevent heat damage to the seedlings. Optional method: Collect soils from selected field sites using soil probe or trowel to sample from upper 10 cm of soil profile; place soil into plastic sample bag, place in cooler for transport to lab. Pass soil through coarse sieve (i.e., 2-mm mesh), dispense into 7.5-cm diam pots, plant seeds of target plant in soil, place in greenhouse allow plants to develop. When desired growth stage is reached, carefully remove intact plants and handle as outlined above. (ii).

Separate root systems from plants by cutting top growth at the “soil line” with a clippers. Soil adhering to roots is vigorously shaken followed by firmly brushing root surfaces with an artists brush to remove visibly attached soil into separate containers and designated as “rhizosphere soil.” Rigorously surface-sterilize the cut root systems: (a). Fill a beaker (250-mL or larger) with ~100mL of 10 % bleach, and 3 other beakers with ca. 100 mL of sterile water. (b). Flame forceps with 95% ethanol in alcohol burner. (c). With flamed forceps, place roots in bleach solution; wash for 2- min. (d). Dab with one non-sterile paper towel, then with sterile paper towel. (e). Wash root fragment in sterile water 3 times – sequentially using the 3 beakers of sterile water.

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(f). Dab surface-sterilized roots with sterile towel to dry. (g). After completely dry, trim off ends of roots with flame-sterilized scissors. At this point, surface-sterilized roots can be immediately processed for plating on selective agar or can be wrapped in sterile paper towels and held in the refrigerator for 1 – 2 days before plating. (iii). Root Processing for Fusarium plating (17): (a). Select root fragments for plating: For young plants, most lateral roots can be used; for older plants (later in the season), use lateral roots from upper half of intact root system. (b). Using flame-sterilized scissors, cut root fragment into 2-cm segments; with flame-sterilized forceps, place up to 8 segments onto agar surface of one agar plate, making sure agar contact is made. In this manner set up replicate plates per plot [four replicates are desirable, therefore, 32 2-cm root segments are required per plot (experimental unit]. (c). Incubate plates on laboratory bench at ambient temperature and light. (iv).

(v).

Examination of plates and Data Recording: (a). Examine plates beginning 5-7 d after incubation. Fusarium colonies are usually white and begin appearing on roots as small cottony spherical colonies. Try to record numbers of colonies per segment per plate before the colonies begin to merge together. (b). Record numbers of colonies per segment per plate. (c). After or during enumeration of Fusarium colonies, individual colonies can be subcultured onto another medium (usually Potato Dextrose Agar [PDA]) for subsequent identification and/or bioassays. This can be done by removing a small amount of mycelia from the aerial portion of the colony on root tissue using a flame-sterilized needle and transferring to PDA, gently placing the removed growth onto the PDA surface. Place 3 “stabs” of the growth per PDA plate to assure growth of the isolate is obtained. These plates can be incubated under ambient conditions. After observed cultures on PDA are uniform and considered individual species (this may require several sub-culturing transfers to fresh PDA) can be characterized using descriptions of cultural and microscopic morphologies (Nelson et al., 1983). Taxonomic identification using this procedure can be confirmed using molecular determinations based on specific gene sequences detected with PCR analyses (28). Cultures can then be maintained in a pure state on PDA stored at 4C and serve as a resource for further testing of allelopathic activity on target plants.

Calculations The total number of colonies recorded for each plate is used to determine “density of root colonization,” expressed as “number of Fusarium colonies per 16 cm of root” for each plate. For overall expression of colonization for plant, soil or treatment comparisons, the values are converted to “Fusarium numbers per 100 cm root” according to: Fusarium colonization per 100 cm root = (Fusarium colonies/16 cm root) X 100


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This can also be expressed as “% root colonization” since based on 100 cm root. Statistical analysis Colonization data can be subjected to analysis of variance and, when F values are significant (P < 0.05), means can be differentiated using a mean separation technique such Fisher’s protected least significant difference (lsd) test at α = 0.05.

3. ASSESSING ACTIVITY OF ALLELOPATHIC MICROORGANISMS Effective screening assays are essential to demonstrate that the isolated microorganisms possess allelopathic activity. The bioassays are presented in a logical sequence beginning with high throughput assays for reducing large culture collections to those cultures with greatest allelopathic potential to be tested in more rigourous, hostspecific screening assays. 3.1. SEEDLING BIOASSAYS FOR RHIZOBACTERIA Bioassays with surrogate, sensitive indicator plant species or phytotoxic-sensitive microbial cultures aid in selection efficiency of highly allelopathic rhizobacteria by providing high throughput methods. Bioassays with specific seedlings in hydroponic culture allows the final selection of most allelopathic cultures on host weeds. 3.1.1. Allelopathic Activity of Rhizobacteria: Isolation, sub-culturing and selection of pure cultures of potential allelopathic bacteria from various plants of origin often results in a collection of 1,000 or more cultures in storage (i.e., 15,18). Thus, rapid initial screening bioassays have been established to aid in limiting the number of isolates eventually screened on target plant species to those that exhibit consistent allelopathic activity on indicator species that are sensitive to a wide range of phytotoxins or allelochemicals produced by allelopathic microorganisms. We have developed a comprehensive screening procedure for allelopathic rhizobacteria to address effective selection for potential allelopathic activity (Fig. 1). Initial assays help to select allelopathic rhizobacteria from the bulk of the total culture collection. Subsequent bioassays of plant seedlings of interest should refine the collection to those isolates that specifically inhibit or suppress the growth based on presumed allelochemical production. When a collection of isolates exhibit the allelopathic activity to host plants in greenhouse and/or growth chamber or field trials, the isolates could simultaneously be cultured for production of suspected allelochemicals released into selective media, which can be extracted and analyzed and characterized (dashed arrow connector in Fig. 1). The following experimental procedures outline bioassays (Fig. 1) for selecting allelopathic rhizobacteria active in soils and their subsequent analyses for presumptive allelochemicals (12,15,18). The indicator technique for antimetabolite toxin reaction toward Escherichia coli by phytopathogenic bacteria (7) has been adapted to detect the potential allelochemical production and increased efficiency of selection of presumptive allelopathic bacteria (15). The lettuce (Lactuca sativa L.) seedling bioassay was developed for preliminary evaluation of phytotoxicity of rhizobacteria based on sensitivity of lettuce in consistently indicating effects of growthinhibiting bacteria (3). Finally, a growth pouch hydroponics system based on plastic bags

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Figure 1. Sequence of bioassays for selection and identification of potential allelopathic rhizobacteria from seedling rhizosphere. The final step involving field testing may be coordinated with extraction and chemical analyses for allelopathic compounds from selective culture of rhizobacterial isolates exhibiting activity at the most rigorous step in the sequence. *Inclusion of E. coli antimetabolite bioassay can be optional if number of isolates is not great and it is preferable to begin with lettuce seedling indicator bioassay. Modified from Stubbs and Kennedy (31)

with germination paper saturated with a nutrient solution, originally developed for assays of legume root nodulation by rhizobia (30), provides an efficient assay to screen the selected test seedlings directly with rhizobacterial isolates of interest (15). Materials and equipment required Lettuce seeds, host plant seeds, Escherichia coli culture (a non-pathogenic strain such as ‘B’ or ‘K12’), 250- and 600-mL beakers, stainless steel tea strainers, sterile paper towels, test tubes, mixer, analytical balance, magnetic stirrer, autoclave, pipettes, multichannel pipette (optional), petri plates, bacteriological loops, bacteriological or dissecting needles, forceps, marking pens, labels, calipers or rulers, Bunsen gas burner or alcohol burner, spectrophotometer, growth pouches (Mega International, Minneapolis, Minnesota, USA) for hydroponic screening; rack for holding growth pouches; pots (7.5-cm diam) for greenhouse

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Reagents and culture media required 0.01M MgSO4 buffer: Dissolve 1.203 g of reagent-grade MgSO4 in 1L of distilled water and store in reagent bottle under ambient conditions; 1.25% Sodium hypochlorite (or 10% commercial bleach); 95% ethanol; sterile de-ionized water; ‘Water agar:’ Dissolve 15 g bacteriological agar in 1 L distilled water, autoclave, dispense into petri plates; S1 agar medium (see Table 1); Pseudomonas minimal salts (PMS) medium (see Table 4); Plant nutrient solution (see Table 5). Table 4. Composition of Pseudomonas minimal salts medium (PMS) (7) Ingredients Glucose NH4H2PO4 (ammonium dihydrogen orthophosphate) KCl MgSO4 . 7H2O Agar Deionized water Adjust pH to 7.0 before autoclaving

Quantity 2.0 g 1.0 g 0.2 g 0.2 g 12 g 1000 mL

Table 5. Plant nutrient solution (30) Stock Solution 1 2 3

Nutrient

Form

MW

g/L

Stock molar conc. (M) 2.0 1.0 0.02 0.5 0.5 0.002 0.004 0.001 0.0004 0.0002 0.0002

Final nutrient concentration 1.0 mM 0.5 mM 10 µM 0.25 mM 0.25 mM 1.0 µM 2.0 µM 0.5 µM 0.2 µM 0.1 µM 0.1 µM

Ca CaCl2 . 2H2O 147.03 294.1 P KH2PO4 136.1 136.1 Fe Fe-citrate 355.04 6.7 Mg MgSO4 . 7H2O 246.5 123.3 K K2SO4 174.06 87.0 Mn MnSO4 . H2O 169.02 0.338 4 B H3BO4 61.84 0.247 Zn ZnSO4 . 7H2O 287.56 0.288 Cu CuSO4 . 5H2O 249.69 0.100 Co CoSO4 . 7H2O 281.12 0.056 Mo Na2MoO2 . 2H2O 241.98 0.048 Preparation: 1. For each 10 L of full-strength nutrient solution, dispense 5.0 mL of each stock solution (1- 4), to 5 L of de-ionized water, then dilute to 10 L. Use 1 N NaOH to adjust final pH of final fullstrength nutrient solution to 6.6- 6.8. 2. For a ‘Complete nutrient solution,’ add KNO3 at 0.05% of the final volume to yield a N concentration of 70 mg/L.

Procedures I. E. coli antimetabolite assay: (i). From E. coli stock cultures, sub-culture by transferring and streaking culture growth using bacteriological loop to fresh PMS agar (Table 4) and incubating at ambient temperature for 48 h.

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(ii).

Suspend culture growth by adding minimal volume of 0.01M MgSO4 buffer to plate and gently rubbing growth off surface with flame-sterilized loop; collect suspended bacterial growth by pipetting into test tube; adjust concentration to 108 cells per mL by reading absorbance of suspension on spectrophotometer at 600 nm wavelength; desired concentration is reached at an optical density (OD) reading of 0.55 -6.0; adjust cell density diluting with additions of 0.01M MgSO4 buffer (24). (iii). Add E. coli suspended cells to molten PMS agar at < 40o C at 1.0 mL cell suspension per 10 mL agar; allow to set up (gel) at ambient temperature. (iv). From the freshly sub-cultured rhizobacteria isolates collected in Experiment 1, remove culture growth using a flame-sterilized bacteriological needle and transfer to E. coli-inoculated PMS agar plate (step iii) by gently stabbing into the agar surface at a minimum of three equally spaced points within a plate. Be sure to flame loop between transfers of different isolates. Incubate at ambient temperature for 48 h. (v). Presumptive allelopathic rhizobacteria will inhibit E. coli indicated by a clear zone devoid of growth around the point of inoculation of rhizobacteria. Record reactions induced by each rhizobacterial isolate as ‘plus’ or ‘minus’ and measure the diameter of the clear zone in mm using a ruler or calipers.

Calculations Relative potential allelopathic activity can be determined by averaging zone diameter (mm) over replicate inoculation points. The relative proportion of rhizobacterial isolates exhibiting presumptive allelochemical production can be expressed as: % Inhibiting E. coli = (No. of E. coli inhibiting isolates/Total isolates)/100 Proportion of inhibitory isolates can also be calculated for each group associated with specific host plants, soils, crop or land management systems, etc. II. Lettuce seedling bioassay using water agar: (i). Rhizobacterial cultures with presumptive allelopathic activity selected in the E. coli antimetabolite assay are cultured on S1 agar for 48 h; cultures are suspended in 0.01M MgSO4 buffer using a bacteriological loop, then transferred to labeled tubes using a pipette; adjust cell density to 106 cells per mL, following procedure given in (ii) for the E. coli antimetabolite assay but attain a final OD of 0.3 of the diluted cell suspension. (ii). Surface-sterilize lettuce seeds by placing seeds into a meshed tea strainer and immersing into 10% bleach contained in a 250- or 600-mL beaker for 1.5 min, followed after removal by immersion in sterile distilled water with shaking for 2 min., removal and re-immersion in 70% ethanol for 1.5 min, followed by rinsing in sterile distilled water at lease two times. Surface-sterilized seeds are placed on sterile paper towels under sterile hood and blotted to remove excess moisture. (iii). Carefully transfer seeds to water agar plates using flame-sterilized forceps and space equidistantly on agar surface to allow germination at ambient temperature overnight (< 24 h incubation).


(iv).

(v).

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Carefully select lettuce seedlings with radicles ≤ 2mm long using flame-sterilized forceps and evenly distribute on water agar plates (20 seedlings per plate); inoculate each seedling by dispensing 30 µL cell suspension of rhizobacterial isolate using either a single or multi-channel pipette. Incubate at 27C in the dark for 48 h. Set up control plates using 0.01M MgSO4 buffer as the inoculant for each seedling. Measure radicle lengths in mm using ruler or calipers, also note the obvious seedling growth inhibition symptoms or injuries including root stunting and tip swelling and necroses.

Observations (a). Three replicate plates for each isolate should be set up. Measure radicle length for each seedling within each plate, obtain plate average radicle length to represent one replicate. (b). Qualitative root injury ratings can be determined by evaluating on a scale of 0 to 4, where 0: No injury evident and 4: Abundant necroses, severe stunting, little or no root hair formation, and seedlings dead or nearly so (14). Calculations Along with direct seedling root measurements, the root growth reduction (%) compared to non-inoculated controls can be determined as under: % Reduced root length = [(Control root length – Isolate root length)/Control root length] X 100

Occasional growth stimulation may occur and can be calculated: % Increased root length = [(Isolate root length – Control root length)/Control root length] X 100

Statistical analysis Bioassay results can be analyzed by ANOVA using a standard design such as completely random; mean comparisons can be conducted using Fisher’s least significant difference test at P = 0.05. III. Growth pouch hydroponics bioassay: Procedure (i). Surface-sterilize seeds of target plant species following protocol outlined in (ii) for lettuce seedling bioassay. (ii). Set up growth pouches in racks, dispense approximately 60 mL of nutrient solution per pouch taking care to completely moisten the germination paper. (iii). Place two or three surface-sterilized seeds per pouch using a flame-sterilized forceps to position the seeds in the upper fold (trough) of the germination paper. (iv). Prepare bacterial suspensions for each rhizobacterial isolate selected for bioassay, following the protocol outlined in (i) for lettuce seedling bioassay; inoculate seeds within the growth pouch with 2 mL of bacterial suspension. (v). Place pouches in growth chamber or under bank of lights with fluorescent and incandescent bulbs on the laboratory bench at 25 – 27C with 12-h light and 12-h dark periods.

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Growth period required for adequately developed roots may vary for different plant species but averages about 14 days, after which root lengths can be measured, root injury rated, and shoot growth harvested for biomass weight determinations.

Observations (a). Five replicate pouches for each isolate should be set up. Measure radicle length for each seedling within each pouch, obtain pouch average radicle length to represent one replicate. (b). Qualitative root injury ratings can be determined by evaluating on a scale of 0 to 4, where 0: No injury evident and 4: Abundant necroses, severe stunting, little or no root hair formation, and seedlings dead or nearly so. (c). Abnormal symptoms of shoots can be noted including stunted leaf development, chlorosis, wilting, etc. (d). Growth response of seedling can be easily tracked through time course measurements of root length directly by observing roots in the transparent growth pouch periodically during the growth period (12). (e). Some seeds (i.e., Arachis hypogeae L.) are too large to be assayed in the growth pouch system; these must be assayed in soil or potting mixture in pots in which seeds are inoculated with bacterial suspension, covered and placed in greenhouse for appropriate growth period (15). (f). If allelopathic effects on root development only are of interest, small seeds of target plants can be assayed directly on water agar following the lettuce seed bioassay procedure from which radicle length and damage ratings can be obtained (5,17). Calculations Along with direct seedling root measurements, the root growth reduction (%) compared to non-inoculated controls can be determined - Please see lettuce seedling bioassay calculations. Statistical analysis Bioassay results can be analyzed by ANOVA using a standard design such as completely random; mean comparisons can be conducted using Fisher’s least significant difference test at P = 0.05. 3.2. SEEDLING BIOASSAYS FOR RHIZOSPHERE FUNGI Owing to morphological differences, rhizosphere fungi are screened in bioassays designed to accommodate optimum growth, while allowing production of allelochemicals involved in plant growth-detrimental activity desired in effective biological control of weeds. Representative protocols developed for screening rhizosphere fungi are outlined below. 3.2.1. Bioassays for Allelopathic Activity of Rhizosphere Fungi: Conventional methods to characterize the phytopathogenic fungi based on application of spore suspensions to seedlings and incubating for development of disease symptoms are generally not applicable to screening for allelopathic fungi. Most allelopathic fungi are necrotrophic or opportunistic pathogens that synthesize toxins (allelochemicals) to damage


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or kill plant cells to derive plant-based nutrients; other fungi interfere with plant physiological processes by producing plant growth regulators including gibberellins and auxins (20). Because allelochemical substances are released during active growth of fungus intimately associated with plants, spore inoculation techniques are inadequate in bioassays since a lag time is required before allelochemicals accumulate to incur a plant response. Therefore, screening directly with fungal preparations in which allelochemicals are actively synthesized or have been collected from previously cultured fungi is preferred (32,33). Fungi are not easily cultured as homogenous cell suspensions that are typical for bacterial cultures. Screening procedures for allelopathic activity by fungi follow a similar scheme as for bacteria but are modified to accommodate production and extracellular release of potential allelochemicals for consistent and even contact with the seedling root or other plant structure. The preliminary screening step using the lettuce seedling indicator bioassay is imposed on pure fungal cultures previously incubated on a rice (Oryza sativa L.) grain medium that induces phytotoxin production (1). The fungal cultures are overlaid with water agar, seeded with germinated lettuce, which are sensitive to fungal-produced allelochemicals diffusing from culture media into the water agar, based largely on microbial bioherbicide assays developed by Heisey et al. (10). The subsequent bioassays on specific plant hosts utilize a culture filtrate from a liquid medium in which fungal isolates were grown for selective production of allelochemicals (32). For this bioassay, stem cuttings of target plants grown uniformly in the greenhouse or growth chamber are placed directly in tubes containing the fungal culture filtrates and observed for development of adverse growth symptoms. Materials and equipment required Lettuce seeds, host plant seeds, 250- and 600-mL beakers, 250-mL Erlenmeyer flasks with foam stoppers; 50-mL centrifuge tubes with screw caps; stainless steel tea strainers, sterile paper towels, glass or disposable test tubes ca. 1.5 X 15 cm; test tube rack, mixer, analytical balance, magnetic stirrer, autoclave, centrifuge; pipettes, petri plates, bacteriological loops, bacteriological or dissecting needles, forceps, cork borer (5- to 7mm diam.); sterile syringe filter membranes (0.45 µm diam); 30- or 50-mL disposable syringes; marking pens, labels, scissors or shears, calipers or rulers, Bunsen gas burner or alcohol burner, pots (7.5-cm diam) for greenhouse Reagents and culture media required 0.01M MgSO4 buffer: Dissolve 1.203 g of reagent-grade MgSO4 in 1L of distilled water and store in reagent bottle under ambient conditions; 1.25% Sodium hypochlorite (or 10% commercial bleach); 95% ethanol; sterile de-ionized water; PDA (see Table 3); Rice agar medium (RAM) (see Table 6); Fries liquid medium (see Table 7). Procedures I. Lettuce seedling bioassay using RAM: (i). Retrieve rhizosphere fungal isolates from storage and subculture onto fresh PDA using flame-sterilized needle. After abundant sporulating mycelial growth attained,

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Table 6. Rice agar medium for preliminary screening of fungal phytotoxicity (1) Ingredients Quantity Rice (long grain) 100 g Distilled water 500 mL Agar 15 g Distilled water 500 mL Preparation: 1. Add rice to 500 mL distilled water, autoclave 20 min, allow to cool, pour into Waring blender and blend at high speed for 3 to 5 min. 2. Mix blended rice suspension with 500 mL distilled water, add agar, autoclave 20 min. Allow to cool, dispense into petri plates. Table 7. Fries medium for fungal phytotoxin production (6) Ingredient Ammonium tartrate Ammonium nitrate (NH4NO3) Magnesium sulfate (MgSO4 . 7H2O) Potassium phosphate, dibasic (K2HPO4) Potassium phosphate, monobasic (KH2PO4) Cupric chloride (CuCl2 . H2O) Molybdic acid (H2MoO4) Manganese chloride (MnCl2. 4H2O) Cobalt chloride (CoCl2. 4H2O) Yeast extract Distilled water Mix contents together in distilled water, autoclave for 20 min.

Quantity 5.0 g 1.0 g 0.5 g 2.6 g 1.3 g 107 mg 34 mg 72 mg 80 mg 1.0 g 1000 mL

use flame-sterilized needle or loop to remove growth and transfer to RAM plates by making a straight streak to obtain a ca. 1-cm wide band of actively growing fungi on one side of agar surface. Incubate the inoculated RAM plates at 28oC for 5 to 7 days. (ii). After incubation, prepare 1.0% water agar, autoclave, allow to cool to ca. 37C; pour ca. 10 mL of the cooled agar over each RAM plate to cover the fungal growth. (iii). Surface-sterilize lettuce seeds in same manner as described for “Lettuce seedling bioassay using water agar.” (iv). After the RAM plates overlain with water agar have set (or gelled), carefully transfer seeds to water agar plates using flame-sterilized forceps and space equidistantly on agar surface beginning with a row directly over the immersed fungal streak followed by parallel rows at equal distances away from streak - this should allow 4- rows with 5- seeds each. Allow seedling to grow at ambient temperature for 48 h incubation). Set up control plates without fungal growth to be seeded. (v). Measure radicle lengths in mm using ruler or calipers; also note obvious seedling growth inhibition symptoms or injuries including root stunting and tip swelling and necroses and note whether presumed allelochemicals diffused throughout plate or if activity is limited within the vicinity of the fungal band.


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Observations (a). Three replicate plates for each isolate should be set up. Measure radicle length for each seedling within each plate, obtain plate average radicle length to represent one replicate. (b). Qualitative root injury ratings can be determined by evaluating on a scale of 0 to 4, where 0 = no injury evident and 4 = abundant necroses, severe stunting, little or no root hair formation, and seedlings dead or nearly so (14). Calculations From the direct seedling root measurements, root growth reduction (%) than noninoculated controls can be determined: % Reduced root length = [(Control root length – Isolate root length)/Control root length] X 100

Occasional growth stimulation may occur and can be calculated: % Increased root length = [(Isolate root length – Control root length)/Control root length] X 100

Statistical analysis Bioassay results can be analyzed by ANOVA using a standard design such as completely random; mean comparisons can be conducted using Fisher’s least significant difference test at P = 0.05. II. Secondary bioassay in fungal culture filtrates: Procedure (i). Grow target plant species in growing medium (i.e., nursery potting mix) in containers placed in the greenhouse. Depending on plant species, two to four weeks may be required to obtain vegetative material for testing. (ii). Prepare fresh PDA cultures of fungi selected for testing based on results of lettuce bioassay. Incubate at ambient conditions for 4 to 5 days. (iii). Remove agar plug using a flame-sterilized cork borer, transfer with flame-sterilized forceps to 50 mL of Fries liquid medium in a 250-mL flask; stopper the flask and incubate statically at 25oC for one to two weeks. (iv). Centrifuge static cultures using 50-mL centrifuge tubes at 5,000 rpm for 10 min; pass supernatant through 0.45 filter membrane using a syringe. Dispense filtrate in 10-mL aliquots into sterile test tubes, 5 per culture. (v). Harvest test plant cuttings by clipping top growth of plants grown in greenhouse to provide at lease five uniform stem cuttings per treatment. Place one cutting in each tube containing culture filtrate or sterile water control. Place tubes containing cuttings held in racks in growth chamber or under bank of lights with fluorescent and incandescent bulbs on the laboratory bench at 25 - 27oC with 12-h light and 12h dark periods. (vi). Examine shoot growth in 48 to 72 h for wilting and chlorosis, and rate for damage or injury to shoot growth.

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Observations (a). Chlorosis and wilting of leaves of cuttings indicate the presence of allelochemicals in the culture filtrate. Even though this bioassay may be considered a “presence or absence” test for allelochemical evidence, it is more rigorous than lettuce indicator bioassays since a host plant(s) is used and the severity of allelopathic effects can quantified using a numerical rating scale as suggested by Yang et al. (32): 0: No damage, 1: Chlorosis on or one or two small browning areas on leaves, 2: Three to 15 brown spots on leaves, 3: Leaves wilted and easily detached and 4: Leaves brown and plants dying or dead. (b). The most allelopathic fungi, based on index values determined under “Calculations”, may be selected for more rigorous field testing (although it is highly recommended that the secondary bioassay be repeated at least once). (c). Field testing entails preparation of bulk inocula of fungal cultures, which can be accommodated often by rapid fungal growth on a grain substrate such as rice, as described by Abbas et al. (1). Such preparations can be delivered to soil with host plant seeds or in vicinity of the growing plant and observe for symptoms as described in the “Procedures” above. Calculations An allelopathic index score [based on disease index determinations, Yang et al. (32)] can be calculated from based on severity ratings collected under “Observations:” [summation of (severity rating X number of plants rated at that value)] Allelopathic index = Total number of plants An index at or < 2.9 is considered resistant; that > 2.9 is considered allelopathic. Statistical analysis Bioassay results can be analyzed by ANOVA using a standard design such as completely random; mean comparisons can be conducted using Fisher’s least significant difference test at P = 0.05.

4. CONCLUSIONS The majority of allelopathy research has focused on effects of allelopathic plants and their allelochemicals on growth of other plants and on specific microbial processes such as nitrification (25), very little work has considered environmental microorganisms as allelopathic agents. The lack of research emphasis in this area is largely due to the general inclusion of these microorganisms with the phytopathogens. However, it is clearly demonstrated in this review and elsewhere (4,13) that allelopathic microorganisms comprise a group separate from typical phytopathogens because they do not follow classical disease cycles involving aggressive infection and colonization of plant tissues. Thus, it is important to understand the effects of allelopathic microorganisms on plant growth and discern these from phytopathogens so that useful information can then be applied in developing effective management strategies in agronomic and environmental


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systems. The procedures described herein are presented as templates for devising custom protocols for the development of standardized bioassays needed for consistent detection and characterization of allelopathic microorganisms. The set of protocols is intended for use by graduate students or researchers new to the allelopathy field, in both developed and less developed countries, yet also serve as a valuable resource to scientists of various levels of expertise, agronomic extension personnel, and farmers. The opportunity for improved bioassays based on the information provided in this review will greatly help in better understanding the role of allelopathic microorganisms in affecting plant growth; lead to development of management practices targeted at this group of microorganisms rather than broad approaches aimed at soilborne phytopathogens; and yield insight on the potential of allelopathic microorganisms as weed growth-suppressing agents.

5. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Abbas, H.K., Boyette, C.D., Hoagland, R.E. and Vesonder, R.F. (1991). Bioherbicidal potential of Fusarium moniliforme and its phytotoxin, fumonisin. Weed Science 39: 673-677. Akkermans, A.D.L., van Elsas, J.D. and de Bruijn, F.J. (1996). Molecular Microbiology Ecology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands. Älstrom, S. (1987). Factors associated with detrimental effects of rhizobacteria on plant growth. Plant and Soil 102: 3-9. Barazani, O. and Friedman, J. (1999). Allelopathic bacteria and their impact on higher plants. Critical Reviews in Plant Science 18: 741-755. Ben-Hammouda, M., Kremer, R.J. and Minor, H.C. (1995). Phytotoxicity of extracts from sorghum plant components on wheat seedlings. Crop Science 35: 1652-1656. Dhingra, O.D. and Sinclair, J.B. (1985). Basic Plant Pathology Methods. CRC Press, Boca Raton, Florida, USA. Gasson, M.J. (1980). Indicator technique for antimetabolic toxin production by phytopathogenic species of Pseudomonas. Applied and Environmental Microbiology 39: 25-29. Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R. (1994). Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, DC, USA. Gould, W.D., Hagedorn, C., Bardinelli, T.R. and Zablotowicz, R.M. (1985). New selective medium for enumeration and recovery of fluorescent pseudomonads from various habitats. Applied and Environmental Microbiology 49: 29-32. Heisey, R.M., DeFrank, J. and Putnam, A.R. (1985). A survey of soil microorganisms for herbicidal activity. In The Chemistry of Allelopathy (Ed., A.C. Thompson), ACS Symposium Series 268: 337-349. American Chemical Society, Washington, DC, USA. Kennedy, A.C. (2005). Rhizosphere. In: Principles and Applications of Soil Microbiology (Eds., D.M. Sylvia, J.J. Fuhrmann, P.G. Hartel and D.A. Zuberer). pp. 242-262. Pearson Prentice Hall, New Jersey, USA. Kim, S.-J. and Kremer, R.J. (2005). Scanning and transmission electron microscopy of root colonization of morningglory (Ipomoea spp.) seedlings by rhizobacteria. Symbiosis 39: 117-124. Kremer, R.J. (2006). The role of allelopathic bacteria in weed management. In: Allelochemicals: Biological Control of Plant Pathogens and Diseases. (Ed., Inderjit), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 533-547. Kremer, R.J., Sasseville, D.N., and Mills, H.A., Jr. (1996). Promotion of phytotoxic bacteria in rhizospheres of leatherleaf fern by Benlate DF. Journal of Plant Nutrition 19: 939-953. Kremer, R.J., Begonia, M.F.T., Stanley, L. and Lanham, E.T. (1990). Characterization of rhizobacteria associated with weed seedlings. Applied and Environmental Microbiology 56: 1649-1655. Krieg, N.R. and Holt, J.G. (1984). Bergey’s Manual of Systematic Bacteriology, vol. 1. The Williams & Wilkins Company, Baltimore, Maryland, USA. Levesque, C.A., Rahe, J.E. and Eaves, D.M. (1993). Fungal colonization of glyphosate-treated seedlings using a new root plating technique. Mycological Research 97: 299-306.

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18. Li, J. and Kremer, R.J. (2000). Rhizobacteria associated with weed seedlings in different cropping systems. Weed Science 48: 734-741. 19. Means, N.E., Kremer, R.J. and Ramsier, C. (2007). Effects of glyphosate and foliar amendments on activity of microorganisms in the soybean rhizosphere. Journal of Environmental Sciences and Health, Part B 42: 125-132. 20. Möbius, N. and Hertweck, C. (2009). Fungal phytotoxins as mediators of virulence. Current Opinion in Plant Biology 12: 390-398. 21. Nash, S.M. and Snyder, W.C. (1962). Quantitative estimations by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopathology 52: 567-572. 22. Nelson, E.B. (2004). Microbial dynamics and interactions in the spermosphere. Annual Review of Phytopathology 42: 271-309. 23. Nelson, P.E., Toussoun, T.A. and Marasas, W.F.O. (1983). Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University, University Park, Pennsylvania, USA. 24. Pepper, I.L. and Gerba, C.L. (2005). Environmental Microbiology: A Laboratory Manual, 2nd edition. Elsevier Academic Press, San Diego, California, USA. 25. Rice, E.L. (1984). Allelopathy. 2nd edition. Academic Press, Inc., Orlando, Florida, USA. pp.434. 26. Schippers, B., Bakker, A.W. and Bakker, P.A.H.M. (1987). Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Annual Review of Phytopathology 25: 339-358. 27. Schroth, M.N. and Hancock, J.G. (1982). Disease-suppressive soil and root-colonizing bacteria. Science 216: 1376-1381. 28. Skovgaard, K., Nireberg, H.I., O’Donnell, K. and Rosendahl, S. (2001). Evolution of Fusarium oxysporum f. sp. vasinfectum races inferred from multigene genealogies. Phytopathology 91: 1231-1237. 29. Smibert, R.M. and N.R. Krieg. (1981). General characterization. In: Manual of Methods for General Bacteriology (Ed., P. Gerhardt). American Society for Microbiology, Washington, D.C., USA, pp. 409-443. 30. Somasegaran, P. and Hoben, H.J. (1985). Methods in Legume-Rhizobium Technology. NifTAL Project, University of Hawaii, Paia, Hawaii, USA. 31. Stubbs, T.L. and Kennedy, A.C. (2012). Microbial weed control and microbial herbicides. In: Herbicides Environmental Impact Studies and Management Approaches (Ed., R. Alvarez-Fernandez). InTech-Open Access Publishers, Rijeka, Croatia, pp. 135-166. 32. Yang, S.-M., Johnson, D.M. and Dowler, W.M. (1990). Pathogenicity of Alternaria angustiovoidea on leafy spurge. Plant Disease 74: 601-604. 33. Yang, S.-M., Dowler, W.M. and Johnson, D.M. (1991). Comparison of methods for selecting fungi pathogenic to leafy spurge. Plant Disease 75: 1201-1203. 34. Zahir, Z.A., Arshad, M. and Frankenberger, W.T., Jr. (2004). Plant growth promoting rhizobacteria: applications and perspectives in agriculture. Advances in Agronomy 81: 97-168.