-
The experiment was carried out in the Microbial Biotechnology and Striga Bioassay Laboratories at National Agricultural Biotechnology Research Center (NABRC), Holeta. NABRC is located at 9°3'N latitude and 38°30'E longitude, 34 km away from Addis Ababa, in the central part of Ethiopia, West Shoa Zone of Oromia Regional State.
Source of experimental materials
Soil sample
-
Soil samples were collected from three sorghum growing fields (sites) in the northern region of Ethiopia during the 2018 main crop season. These were Artuma Fursi district in Oromia zone of Amhara region (site 1), Kewet district in Semien Shoa Zone of Amhara region (site 2), and Qaftay Humera in the district West Tigray zone of Tigray region (site 3) with medium, low and high Striga infestation, respectively (Fig. 1). At each site, the soil samples were collected from four random spots in four quadrants after locating the fields using Global Positioning System (GPS) coordinates and recording the altitude for each site (Supplemental Table S1). Soil samples were collected using a sterile shovel at a depth of 20−30 cm and put into a labeled clean plastic bag and transported to the greenhouse facility at National Agricultural Biotechnology Research Center, Holeta.
Sorghum seeds
-
Seeds of different sorghum germplasm that are known to be Striga susceptible, Striga resistant, drought tolerant, widely used, released, and local land races were used in this study (Table 1). The seeds were stored at the National Agricultural Biotechnology Research Center cold room by the national Integrated Striga Control (ISC) project under Ethiopian Institute of Agricultural Research.
Table 1. Sorghum genotype selection for greenhouse planting and isolation of bacteria.
Code Name Source Character Selection criteria G1 ETSL101847 Tigray Local land race Land race and widely used G2 ETWS 90754 Amhara Wild type Wild type G3 ETWS 91242 Beneshangul Wild type Wild type G4 Framida Purdue University Striga resistance Striga resistant and widely used G5 ETSL100046 Land race LGS Land race and LGS G6 ETSL101853 Land race HGS Land race, widely used and HGS G7 Misikir MI_Drought_Score Drought tolerant Drought tolerant G8 S35 ICRISAT Stay green Stay green or Drought tolerant G9 Shanqui red China Striga susceptible variety HGS and Striga susceptible variety G10 SR5-Ribka IBC Striga resistant and fusarium compatibility Striga resistant and fusarium compatibility G11 SRN39 Purdue University Striga resistance Striga resistant and widely used G12 Teshale ICRISAT Best released susceptible varieties Widely used LGS = low germination stimulant; HGS = high germination stimulant, G = genotype. Striga seeds
-
S. hermonthica seeds used for this experiment were collected from S. hermonthica-infested sorghum in farmers' fields in Ethiopia, Tigray Region, Central Zone Abergele District Titay Hagum Kebele during 2018 main cropping season (altitude: 1,466 m; latitude: 13.25'51.8'' East; longitude: 38.59'50.3'' North).
Planting sorghum in the soil in the greenhouse
-
Seeds of sorghum that were well matured and with good morphological characteristics were selected and surface sterilized in 1.5% bleach for 30 min. The seeds were then allowed to germinate for about 30 h in an incubator set to 30 °C. Seedlings were transferred to a pot and grown on the soils collected from various sorghum growing sites in greenhouses. The management including watering and weeding was made accordingly until it was set three leaves ready for harvesting rhizosphere soil samples.
Rhizosphere soil collection
-
Rhizosphere soil sample collection was made following the method described previously[17]. After setting the third leaf, the sorghum was uprooted and vigorously shaken by hand for 5 min until non-adhering soil was completely removed. Rhizosphere soil was collected by removing the sorghum's soil parts with a sterile blade and shaking the roots for 10 min in 50 mL falcon tubes containing 35 mL sterile distilled water to remove the adhering soil. The soil suspensions were then incubated to homogenize the soil content on a shaker (300 rpm, 90 min, and 25 °C) before being centrifuged at room temperature for 10 min to concentrate soil particles in the pellet.
Rhizosphere bacteria isolation
-
Rhizosphere bacteria were isolated by serial dilution technique. One gram of each soil pellet was suspended, each in 90 mL sterile distilled water in a 50 mL falcon tubes and mixed thoroughly overnight using a mechanical shaker at 110 rpm, until completely dispersed. Then a 100 µl aliquot was transferred with sterile pipettes to 9 mL sterile 0.85% saline solution in a test tube. A serial dilution (up to 10–8) was prepared. From each 10–4, 10–5, and 10–6 serial dilutions, 0.1 mL of an aliquot was spread on nutrient agar on Petri-dishes (90 mm), for each dilution in triplicate. Plates were incubated at 28 °C for 24 hours. Representative types of bacterial colonies were further purified by sub-culturing on fresh medium and used for downstream work or stored in 35% glycerol at −80 °C[12,17,18].
In vitro screening of rhizoabcterial isolates for their biocontrol traits
-
Rhizobacteria isolates were first screened for production of HCN followed by screening HCN positive isolates for IAA production. Common producers of HCN and IAA isolates were selected for further evaluation of their effects on S. hermonthica germination inhibition in vitro.
Hydrogen cyanide (HCN) production
-
HCN production by the bacterial isolates was tested qualitatively using the method previously reported[19] with slight modification in incubation period. The bacterial isolate cultures were streaked on Trypto Soya Agar amended with 4.4 g/L glycine. Whatman filter papers were sterilized and soaked in 2% sodium carbonate in 0.5% picric acid solution was placed in the top of each plate. Plates were sealed with parafilm and incubated for 4 days at 28 °C. The change in the color of Whatman filter paper from yellow to light brown, brown or reddish brown was observed as an indication of weak, moderate or strongly hydrogen cyanide producers, respectively.
Indole acetic acid (IAA) production
-
The ability to produce IAA of the isolate was detected from the culture of the bacterial isolates following the procedure described previously[20]. Briefly, pure colonies from a 24-h culture were inoculated into nutrient broth supplemented with 2 % tryptophan and in the absence of tryptophan (control), and incubated at 28 °C for 48 h. Five milliliter culture was removed from each tube and centrifuged at 12,000x g for 15 min. Two milliliter aliquot of the supernatant was transferred to a fresh tube. This was then treated with 2 mL salkowsky reagent (1 mL 0.5 M FeCl in 50 mL HClO4) and incubated at room temperature for 25 min. Development of pink color indicates positive result for IAA production.
In vitro evaluation of the effects of selected isolates on Striga germination
-
Each common HCN and IAA producing isolates was evaluated for their Striga germination and seed decay activity in Striga bioassay laboratory using Agar Gel Assay (AGA) and Whatman filter paper. To do this, S. hermonthica seed germination test was conducted to determine its viability prior to use in vitro evaluation of the effects of rhizobacterial isolates on the seed in the absence and presence of susceptible hosts. However, the seed had to be exposed to the right environmental conditions for the optimum period of time to break dormancy and ready for germination. Hence, the Striga seed was conditioned by incubating at 29 °C for 10−14 d. In each case, S. hermonthica seed was treated with each isolate and germination percentage computed to see the germination inhibitory activity of the isolates[21, 22].
Determination of germination percentage of S. hermonthica
-
S. hermonthica seed surface sterilization and preconditioning was made according to the protocol previously reported[22]. First, seeds were surface sterilized in 75% ethanol under a hood in a 50 mL flask for 2 min and rinsed three times with sterile double distilled water. This was followed by washing the seed with activated metricide (fungicide) for 2 min and rinsed three times with sterile double distilled water. Finally, 14.5 mL ddH2O and 1.5 mL of Benomyl solution (conditioning solution) was added to the flask. The flask was wrapped with aluminum foil and incubated at 30 °C for 10 d for preconditioning.
After 10 d of preconditioning, about hundred sterilized S. hermonthica seeds were transferred into a sterile glass fiber disc on a Petri plate lined with moist Whatman filter paper. Three glass fiber discs on each plate containing preconditioned S. hermonthica seeds were germinated by adding 20 µl of 0.1 ppm GR24 and incubated for 2 d at 28 °C[23]. Negative controls containing preconditioned S. hermonthica seeds were added to sterile distilled water. The numbers of germinated and non-germinated S. hermonthica seeds were counted using a binocular microscope fitted with a digital camera (Power Shot A640, Canon Inc., China). Germination percentage of Striga was determined by counting the total number of seeds on each disc and germinated Striga[22,16].
Evaluation of the effects of the isolates on S. hermonthica seed germination in the absence of host plants
-
The isolates were evaluated for their ability to reduce Striga germination by using GR-24, a synthetic germination stimulant. Striga seed surface sterilization and preconditioning was done as described in a previous section. About 100 preconditioned Striga seeds were added to glass fiber disc placed in Petri plate lined with double sterile filter papers and moistened with 3 mL of sterile ddH2O. The experiment was replicated 3 times each (three glass fiber discs per Petri plate). The seeds on the disc were treated with 100 µl of three days old bacterial suspensions in broth. In the control treatment, blank broth was added to discs containing preconditioned S. hermonthica seeds. The Petri plates were sealed with parafilm and incubated at 30 °C in the dark for 48 h.
After 48 h, 20 µl of 0.1 ppm GR24 was added to keep the germination uniform except for the effect of isolates and further incubated overnight at 29 °C. The number of total S. hermonthica seeds and the number of germinated/ inhibited per replicate was recorded under a stereomicroscope fitted with a camera[16]. Germination percentage for each replicate was calculated using the formula described previously[22].
$ \begin{aligned}&\rm Germination\;percentage\,(GP)=\rm\dfrac{\rm No.\; of\; germinated\; Striga \; seeds}{Total\;no.\;of\;Striga\;seeds}\times 100\end{aligned} $ Evaluation of the effects of the selected isolates on S. hermonthica seed in the presence of susceptible host
-
Striga conditioning was made as explained in the above section, but in this case, it was embedded in agar (bacto agar) solution after 5 d. By using a glass Pasteur pipette, a drop of preconditioned Striga seeds were added to the center bottom of a sterile plate in the conditioning flask. The seeds were treated with 0.5 mL of three days old of bacterial suspension in broth and kept for 30 min. In the control treatment, seeds were treated with blank nutrient broth media. Each treatment was replicated three times and arranged in RCD in an incubator at 30 °C. 0.7% (g/l) agar solution was prepared and autoclaved for 15 min and then allowed to cool in containment room water bath to 50 °C. The liquid agar was directly poured over the striga under hood until the agar reaches the sides of the plate and the striga seeds were distributed evenly across the plate. Plates were allowed to cool for 10 min before covering and placed in a dark at 30 °C in incubator for 10 d from the conditioning start date of the Striga seed.
Simultaneously with Striga conditioning, surface sterilization was made on susceptible sorghum seeds called Teshale using 1.5% bleach (containing a drop of Tween-20) and agitated three times for 30 min. The bleach solution was then poured off and rinsed two times with sterile ddH2O. The seeds were then soaked overnight to imbibe in 5 mL of a 5% (w/v) Captain solution. Next day, the Captain slurry was poured off under a laminar flow hood and rinsed with 5 mL sddH2O. Then, the seeds and water were poured into labeled sterile Petri dishes, each containing two Whatman filter paper (90 mm) circles and incubated until radicles emerged[22].
Next day, the germinated sorghum was gently picked up with sterile forceps and planted 1 cm from the edge of the plate pointing to the center of the plate in agar in which the Striga seeds were already embedded. The plates were incubated at 30 °C in an incubator where they remained for 3 d.
After 3 d, a 2 cm × 2.5 cm area measured was made along the main sorghum root 2 cm from the kernel at the back side of the agar plate using a thick water-resistant marker pen. This area is with high probability of Striga seeds coming into contact with sorghum root exudates. Total and germinated Striga seeds in each area were counted under a stereomicroscope and germination percentage computed using the methods described in the previous section[24,15].
Morphological and biochemical characterization and identification of the most effective rhizobacteria isolates
-
The most efficient bacterial isolates with production of HCN and IAA and corresponding inhibitor of S. hermonthica indicated by low mean germination percentage were selected. These isolates were morphologically and biochemically characterized using the method described previously[25,26] as described below.
Morphological characterization
-
The efficient bacterial isolates were characterized by growing on nutrient plate for 24 h at 28 °C. Best candidate of bacterial isolates were observed under stereomicroscope for colony size, shape, color, arrangement and gram reaction. For Gram staining, slide was cleaned with detergent and marked by codes of isolates. With the help of sterile wire loop, single colony of bacterial culture was made on clean glass slide and air dried and heat fixed. Then smear was covered with crystal violet for 1 min and slide was washed with drop of distilled water. Smear was covered with 2 drops of iodine solution for 30 s and slide was washed with alcohol and then distilled water. The smear was covered with 1 drop of safranin for 1min and then washed by distilled water, air dried and observed under microscope.
Biochemical characterization
-
Each efficient bacterial isolate was tested for sugar utilization, production of methyl red, indole and catalase. This would help to identify the isolates at genus level.
Sugar utilization test
-
The ability of the isolates to utilize carbohydrate sugars as a sole carbon source was determined in broth media containing specific sugar (glucose, fructose and sucrose) and Bromocrsol purple (0.4 g/l). A 96 deep well ELISA plate filled with 1 mL broth was inoculated by 0.1 mL of fresh culture in triplicate including control. The culture was incubated at 28 °C for 24 h and observed for the formation of yellow color as positive results.
Methyl red test
-
Broth containing (5 g of each Peptone, Glucose, Potassium phosphate and 1,000 ml distilled water; pH = 7) was prepared and steam sterilized using autoclave. In test tubes, 1.5 ml of the broth was poured and each was inoculated with test organism, and then incubated at 28 °C for 48 h. Four drops of methyl red indicator was added to each tube and gently shaken for 30 s. The tubes were kept for 15 min and observed for color change (where, positive test = bright red and negative test = yellow to orange)
Catalase test
-
The nutrient agar slants were inoculated with test isolates. An inoculated nutrient agar slant was kept as control. The cultures were incubated at 28 °C for 24 h. A loop full of bacterial culture was kept on a clean slide with the isolate label. A drop of 3% hydrogen peroxide was added on a slide. The culture was then observed for the gas bubble formation.
Statistical analysis
-
All the experimental units were arranged in CRD. Data on effects of selected isolates in S. hermonthica seeds germination was recorded. R software version 3.5.3 was used to perform analysis of variance (ANOVA) for all measured data. Tukey's test was used to compare and separate the means for significance level at 5%.
-
A total of 117 bacteria were isolated from rhizosphere of 12 sorghum varieties grown on soil collected from three different sorghum growing regions in Ethiopia (Supplemental Table S2). The isolates were first tested for their qualitative hydrogen cyanide production on a nutrient agar plate. From these, only 47 (40.2%) of the isolates were found to produce HCN with different levels (low producers, medium producers and strong producers).
Forty seven isolates capable of producing HCN were again tested for IAA production, another weed suppressive trait of rhizobacteria. Accordingly, six (12.8%) isolates were strong producers, 9 (19%) were moderate producers and 7 (14.9%) were low or weak producers and 25 (53.2%) were not producers of IAA at all.
From both test, 21 isolates were common producers of HCN and IAA. These include E19G1, E19G3, E19G6a, E19G9, E19G11a, E19G6b, E19G10, E19B, E19G7, E19G11b, E19G12, E29G2a, E29G11, E29G2b, E29G9, E29G7, E40G1a, E40G5, E40G1b, E40G10, and E40G12. These isolates were selected for further evaluation of their effects on S. hermonthica germination inhibition in vitro. Majority of the selected isolates did vary in their HCN and IAA production abilities. Some strong producers of HCN were comparably moderate and weak producers of IAA and vice versa. A few isolates, however, showed similarity in their HCN and IAA production. Two isolates, namely E19G12 and E29G7 were strong producers of both HCN and IAA in common.
Determination of germination percentage of S. hermonthica
-
In this study, the germination test for S. hermonthica resulted in 63% germination upon conditioning the seeds for 10 d and treating with GR-24, a synthetic germination stimulant.
In vitro evaluation of the effects of selected isolates on GR-24 induced germination of S. hermonthica
-
In this study, bacterial isolates were evaluated for their effects on Striga germination/inhibition in vitro. The results of the assay showed that significant differences (p < 0.001) were observed between some isolates on the effects of rhizobacteria isolates on GR-24 induced S. hermonthica germination in the filter paper (Figs 2 & 3). Regardless of considerable variation in their inhibition effects, all isolates showed a significant reduction in germination percentage compared to the control (broth treatment). But, the extent of germination inhibition varies from 9 to 59.7 mean germination percentage.
Figure 2.
Effect of rhizobacteria isolates on GR-24 induced S. hermonthica germination in filter paper assay. Values are means of combined data of three replicates each. Means followed by the same letter are not statistically different at p ≤ 0.05 according to the Tukey-test.
Evaluation of the effects of selected isolates on S. hermonthica seed germination in the presence of susceptible host
-
This was an activity done as an alternative to the greenhouse evaluation to see whether there are similar or different trends compared to the evaluation using GR-24 as a stimulant. The study indicated that the germination of Striga in the presence of host plant was lower than that of GR-24 induced germination in all treatments. There was significant difference (p < 0.001) in mean germination percentage among isolates (Figs 4 & 5).
Figure 4.
Effects of rhizobacterial isolates on S. hermonthica seed germination in the presence of susceptible host plant.Values are means of combined data of three replicates each. Means followed by the same letter are not statistically different at p ≤ 0.05 according to the Tukey-test.
A few isolates showed increased germination of S. hermonthica seeds, while many of them showed a significant suppression of S. hermonthica seed germination (p < 0.001) compared to control treatment (broth). The highest germination percentage (27%) were recorded in the control (blank broth treated seeds), followed by isolates E19G9 (24%) and E40G5 (20%). Treatment of S. hermonthica seeds with isolates E19G12, E29G2b, and E19G10 resulted in the lowest S. hermonthica seed germination of 0% 1%, and 2.7% respectively, which were significantly lower than any of the treatment (Fig. 4). Germination inhibition followed an almost similar pattern to the treatments without the presence of host plant except that treatment with the synthetic stimulant GR-24 caused an elevated germination percentage compared to treatment in the presence of host plant sorghum.
Regarding mean germination percentage in the absence of host plant, eight isolates, namely, E19G6a, E19G9, E19G6b, E19G10, E19B, E19G12, E29G2b, and E29G7 showed significant inhibition of S. hermonthica germination as indicated by low mean germination percentage, 16, 10, 26, 9, 29.7, 14, 12 and 18, respectively. Similarly, some isolates that have indicated high Striga germination inhibition in the absence of host plants also showed reduced germination in the presence of host plants although not correspondently or in the same pattern. There was also no consistent pattern in all isolates and parameters evaluated in association with various sorghum genotypes from where they have been isolated and the three soil types. But, majority of the bacterial isolated from the soil of low Striga infested site (E29) potentially inhibited Striga germination in the absence of host plant.
Morphological and biochemical characterization and identification of the most effective rhizobacteria isolates
-
Finally, upon in vitro evaluation, eight efficient rhizosphere bacteria isolates with different Striga suppressive effects were further morphologically and biochemically characterized for gram reaction, colony color, size, shape, margin, elevation, sugar utilization ability, catalase and methyl red test. Accordingly, 6 (75 % ) of the rhizosphere bacteria inhibiting Striga germination were found to be gram negative, 2 (25%) gram positive, 4 (50%) glucose positive, 7 (87.5% ) fructose positive, 2 (25%) sucrose positive, 6 (75%) methyl red positive, 6 (75%) catalase positive (Tables 2 & 3).
Table 2. Morphological characterization and identification of the most effective rhizobacteria isolates.
Isolates Morphological characterization Pigment Shape Size Elevation Margin Gram staining E19G6a White Circular Medium Raised Entire – E19G9 White Circular Medium Raised Entire – E19G6b Brown Circular Medium Raised Entire + E19G10 White Circular Medium Raised Entire – E19B Brown Irregular Large Raised Flat – E19G12 Brown Circular Medium Raised Entire + E29G2a White Irregular Large raised Mucoid – E29G7 White Circular Medium Raised Entire – + = Positive for a given test, – = Negative for a given test under consideration. Table 3. Biochemical characterization of the most effective rhizobacteria isolates.
Isolates Biochemical tests Glucose Fructose Sucrose Catalase Methyl red Tentative identification E19G6a + + – + + Pseudomonas sp. E19G9 – – – + + Pseudomonas sp. E19G6b – + – + + Bacillus sp. E19G10 + + + – – Klebsiella sp. E19B + + – + + Pseudomonas sp. E19G12 – + – + + Bacillus sp. E29G2a – + + – – Entrobacter sp. E29G7 + + – + + Pseudomonas sp. – = no sugar utilization, catalase and methyl red negative, + = sugar utilization, catalase and methyl red positive. Based on the comparative analysis of various morphological and biochemical characteristics, the bacterial isolates are identified to fall under four genera: Bacillus, Pseudomonas, Enterobacter and Klebsiella. Among the bacterial genera, four were Pseudomonas, two Bacillus, one Enterobacter and one Klebsiella (Table 3).
-
We thank the National Agricultural Biotechnology Research Center and its staff for helping with the provision of facilities during the study. We are also grateful to the Ethiopian Institute of Agricultural Research for facilitating this research from sample collection to laboratory analysis of the evaluation of the isolates.
-
About this article
Cite this article
Tulu UT, Haileselassie T, Abera S, Tessema T. 2024. Screening and identification of potential Striga [Striga hermonthica (Del.)] suppressing rhizobacteria associated with Sorghum [Sorghum bicolor (L.) Moench] in Northern Ethiopia. Technology in Agronomy 4: e013 doi: 10.48130/tia-0024-0008
Screening and identification of potential Striga [Striga hermonthica (Del.)] suppressing rhizobacteria associated with Sorghum [Sorghum bicolor (L.) Moench] in Northern Ethiopia
- Received: 25 December 2023
- Accepted: 07 April 2024
- Published online: 04 June 2024
Abstract: Sorghum (Sorghum bicolor (L.) Moench) is one of the globally important cereal crops well adapted to Sub-Saharan Africa (SSA) agro-ecology. However, the productivity of sorghum is hindered by both abiotic and biotic factors including drought, Striga, insect pests, poor soil fertility, and diseases. Among the constraints, Striga (genus), also called witch weed, is the most important production problem in the area. Although there have been various control methods practiced for years, none of these have been practically effective in eradicating Striga, neither are they easily accessible for small holder farmers, while some are also not environmentally friendly. Therefore, this study was designed with the objective of identifying potential Striga suppressing rhizobacteria associated with sorghum. Treatment of S. hermonthica seeds with isolates E19G12, E29G2b and E19G10 resulted in the lowest S. hermonthica seed germination of 0%, 1%, and 2.7% respectively, which were significantly lower than any of the treatments. Mean germination percentage ranged from 9 to 59.7 and 0 to 27 in the absence and presence of host plants, respectively. The results showed a statistically significant germination inhibition (p < 0.001). Finally, the most effective isolates were shortlisted, E19G6a, E19G9, E19G6b, E19G10, E19B, E19G12, E29G2a, and E29G7 were morphologically and biochemically identified to belong to the genera of Pseudomonas, Klebssiella, Bacillus and Entrobacter. The results of the study demonstrated the existence of promising soil-borne bacteria that could be exploited as bioherbicides to control Striga infestation on sorghum provided that broader samples from various parts of the country are explored.
-
Key words:
- Bioherbicides /
- Germination inhibition /
- Rhizosphere /
- Striga infestation /
- Susceptible host