1 In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 11 (3), pp. 001-004, March, 2023. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Evaluation of some bacterial isolates as germination stimulants of Striga hermonthica O. O. Babalola1* , D. K. Berner2 and N. A. Amusa3 1 Department of Microbiology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria 2 Plant Health Management Division, International Institute of Tropical Agriculture, Ibadan, Nigeria. Present address: United States Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Frederick, MD 21702, USA. 3 Department of Plant Science and Applied Zoology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria Accepted 09 December, 2022 To investigate the role of Pseudomonas sp., Klebsiella oxytoca and Enterobacter sakazakii in Striga hermonthica seed germination, we have used two varieties of sorghum to test over time for the selected bacteria, using GR-24, a synthetic strigol analogue as a standard and water as a check. Sorghum seeds coated with a mixture of bacterial cells (5 x 10 7 cfu ml -1 ) from 24 h old cultures were planted in pasteurized potted soil, which was infested with 0.05 g (about 3000) viable S. hermonthica seed per pot 14 d before sowing. Results have indicated that the bacterial isolates could stimulate S. hermonthica germination in the laboratory and in the screen-house. In vitro, only Pseudomonas sp. 4MKS8 gave significant stimulation of S. hermonthica seed at 5% level of probability as compared to the water check. In the screen-house, the four bacterial isolates stimulated significant germination of S. hermonthica. Key words: Striga hermonthica, Pseudomonas sp., Klebsiella oxytoca, Enterobacter sakazakii, germination stimulant, sorghum. INTRODUCTION In the sub-Saharan Africa, it is a common occurrence for the plants to be attacked by one pathogen or another. One of such pathogens attacking plant is Striga, a genus of obligate root-parasitic flowering plant belonging to the family Scrophulariaceae. Striga species constitutes a big constraint to sustainable development and poverty alle- viation. The main graminaceous crops attacked by Striga hermonthica include maize, sorghum, pearl millet, finger millet, upland rice and sugar cane (Berner et al., 1995). In addition to the conditions necessary for the germination of any seed, Striga requires the presence of a stimulant. Today many farmers lack an understanding of its biology and parasitic nature and so cannot manage Striga in the field. Researchers have come to understand that no single methodology will be completely effective in elimina- ting Striga infestations in farmer’s fields. Meanwhile research efforts by scientists employ multidisciplinary approach in the combat of Striga. Microorganisms are in- *Corresponding author. E-mail: olubukola_babalola@yahoo.com. Tel: +2348058871359. creasingly being considered as control agents for Striga (Babalola et al., 2002; Babalola et al., 2004) . Among the micro- organisms colonising the root surface are bacteria of the genus Pseudomonas (Babalola, 2002) of which fluorescent pseudomonads are of special importance (Vancura, 1980). The composition of the microbial association on the root surface can be modified by the introduction of bacterial cells on the surface of seed, roots or tubers. Plant growth-promoting rhizosphere bacteria possess a number of physiological properties, which are important to colonisation of the surface of plant roots and enhancing the growth and health of plants (Vancura and Kunc, 1987). Several methods are avai- lable to control Striga including cultural practices, appli- cation of fertilizers, herbicides, and germination stimu- lants, and planting resistant crop varieties. However, none of these methods alone provides acceptable levels of control (Abbasher et al., 1996) . Therefore, the object- ive of this study was to increase suicidal germination of Striga by using bacteria inoculants in mixed cropping of cowpea with sorghum. 2 MATERIALS AND METHODS Isolation of rhizosphere bacteria Root samples of potted maize and sorghum were shaken vigorously by hand to remove loosely adhering soil. Bacteria from the region of soil neighbouring the root and influenced by it (exorhizosphere, Klyuchnikov and Kozhevin, 1991) and intercellular spaces accessible to bacteria (endorhizosphere) were obtained from 1 g firmly adhering soil or macerated roots (Mawdsley and Burns, 1994). 1 g of firmly adhering soil was shaken in 100 ml of 0.01 M phosphate buffer solution (PBS) solution (0.88% (w/v) NaCl, 2.9 mM KH2PO4, 7.1 mM K2HPO4, pH 7.2) for 2 h. on a rotary shaker (Orbit shaker bath, U.S. Patent No: Des. 288.600, U.S.A) at 200 rpm. Surface sterilisation effected by immersion in 70% ethanol for 30 s. and in 4% solution of sodium hypochlorite (NaOCl) for 3 min. was carried out on the root sample to ensure that the isolates to be cultured were from the endorhizosphere. These were transferred to the laminar flow hood (ENVIRCO, Environmental Air Control, Inc.). Subsequently, serial dilutions (up to 10 –8 ) were prepared, beginning with a 1 ml aliquot of the stock suspension. One ml of the suspensions from the dilution levels 10 -3 to 10 -8 was dispensed in separate 9 cm diameter sterilised Petri-dishes containing King’s medium B (KB) in triplicate. The plates were swirled gently in a clockwise motion to mix the suspension over the agar. After incubation for 24 h at 28 o C, representative types of bacterial colonies were further purified on KB and stored in 35% glycerol at -80 o C. One hundred and eighty isolates of rhizosphere bacteria were screened in preliminary laboratory tests for in vitro stimulation of Striga. hermonthica. Four potential isolates, (isolates in which S. hermonthica germination was observed) were further tested in the screen-house by seed coating. S. hermonthica germination test Striga seed viability was carried out according to the method of Eplee and Norris, (1987). Variability in infection, which may be due to differences in age and quality of inoculum, hosts, growth conditions, and levels of infestation was minimised by using the same seed stock of S. hermonthica, Bida 97. These seed were collected from the same site (Bida, Latitude 9 0 05’, longitude 6 0 01’) and host (sorghum, cultivar unknown) in the same year (1997), hereafter referred to as S. hermonthica BD 97. Besides, the seeds were subjected to uniform conditioning. The S. hermonthica seeds were characterised by a high germinability response to the synthetic strigol analogue, GR-24. All glassware and forceps were sterilised prior to pre-germination tests. The parasite seed were surface- disinfected for 5 min. in a 1% NaOCl. Floating seeds were discarded. The seeds were then thoroughly rinsed in three changes of sterile de-ionised water inside a funnel lined with Whatmann no. 2 filter paper (Whatman ® Whatman Int. Ltd, maidstone, England), air- dried over a clean surface, and stored in glass vials at 28 o C. To condition, the dried S. hermonthica seeds were sprinkled on 3 mm diameter glass-fiber (GF/C, Whatman ® Whatman Int. Ltd, maidstone, England) discs (30 - 40 seed/disc) that were placed on moistened filter paper in Petri-dishes. These were placed in an incubator (Gallenkamp, UK) in the dark at 28 O C for 14 d. After incubation, the glass-fiber discs with the conditioned S. hermonthica seeds were removed from the Petri-dishes and placed in clean 9 cm diameter Petri-dishes. The discs were placed around a 2 cm diameter sterilised film canister filled with tempered Potato dextrose agar (PDA) (Difco) medium, centred in the Petri-dish, on freshly moistened filter paper. The discs were arranged in four radii forming a “cross” radiating from the film canister. Each line had four discs, with the first disk in each line touching the central canister. Pure inoculums of the bacteria to be tested were aseptically streaked on the PDA. In negative controls, 300 µl of sterile deionis- ed water was pipetted over uninoculated PDA as a substitute for ba- cterial inoculum. In the positive control, one parts per million (1 ppm) GR-24 was used. These were then incubated at 28 O C for 3 d and the percentage germination of S. hermonthica seeds on each disk was counted. Average percentage germination for each line of disks in each dish was calculated. Screen-house experiments Plantings were done in screen-house potted soil infested with S. hermonthica. Soil sterilisation was done at 100 0 C for 4 h in Lindig (boiler) machine (Model ‘’SF’’ Burner, MP1192, R.W. Beckett Corp. Elyria, Ohio, U.S.A). For the infested soil, S. hermonthica was added at a concentration of 500 seeds per 4 kg potted soil. The experimental site was at the International Institute of Tropical Agriculture (IITA) Ibadan in Oyo State, Latitude 7 0 43’N longitude 3 0 9’E Nigeria. Seeds of susceptible sorghum (varieties CK60B and Mokwa local) obtained from collections maintained at IITA were treated with bacterial isolates by pelleting with a mixture of bacterial cells from 24 h. old culture (5 x 10 7 cfu ml -1 that was suspended in 1% methylcellulose (mc)). MC is effective as a bacterial preservative and does not significantly influence seed germination (Suslow and Schroth, 1981). Finely ground vermiculite, capable of passing through 300 mm mesh sieve was wrapped in aluminum foil and sterilised in the autoclave at 121 o C for 15 min. The vermiculite added to freshly inoculated wet seed in a beaker and mixed quickly for 2 min. until seed were evenly coated. The coated seed appeared as off-white and were allowed to dry overnight by spreading on a clean surface prior to sowing in sterilised potted soil infested with S. hermonthica BD 97. The sorghum seeds were sown immediately; and where this could not be done, the seeds were stored for not more than 3 weeks at low temperatures (4 O C) in a refrigerator. Seed, which were pelleted with a mixture, which did not contain bacterial cells (mc + powdered vermiculite), served as control. Statistical analysis Means of percentage germination were calculated for each Petri- dish (average of four lines) of each bacterium in each test. The data were analyzed using the UNIVARIATE procedure of SAS (Statistical Analysis System, SAS Institute, Cary, NC) to generate raw means and standard error. Test of the null hypothesis that the germination stimulated by each isolate equaled zero was done by employing the UNIVARIATE procedure. Analysis of variance for each isolate was done by the General Linear Model procedure of SAS. Experiments were repeated four times with four replicates each. RESULTS Isolation of rhizosphere bacteria Forty isolates of bacteria were obtained from the rhizosphere of maize variety 8338-1 and 140 from rhizosphere of two cultivars of sorghum (CK60B and Mokwa local) grown in the screen- house at IITA Ibadan. Isolations were made from the exorhizosphere and endorhizosphere at biweekly intervals by destructive sampling. The bacterial isolates were evaluated for capability to stimulate germination of S. hermonthica BD 97. In a series of laboratory bioassays, a fluctuating (irregular) germination trend was observed in the S. hermonthica seeds as caused by the bacterial isolates. Some of the isolates were tentatively identified as fluorescent pseudomonads based on their water-soluble, 3 Table 1. Germination of S. hermonthica seeds from a population of sorghum plants from Bida, Nigeria, stimulated by selected rhizosphere bacteria. Bacteria % germination of S. hermonthica seeds x mean y Pr>t Dish 1 Dish 2 Dish 3 Dish 4 Pseudomonas 10MKR4 1.751 0.001 0.001 0.001 0.4378 0.7591 z K. oxytoca 10MKR7 2.351 3.701 0.001 3.381 2.3586 0.1066 Pseudomonas 4MKS8 10.611 0.001 0.001 1.591 3.0510 0.0398 Ent. sakazakii 8MR5 1.211 0.001 2.061 0.001 0.8185 0.5675 GR-24 72.801 57.201 68.101 61.891 64.9985 0.0001 Water 0.001 0.001 0.001 0.001 0.0010 1.0000 Std Error 1.41 LSD 5% 2.59 Prob. of F 0.0001 Coef. of variation 37.25 N = 16 for each day GR-24 = +ve control (Strigol-analogues, a synthetic germination stimulant) Water = -ve control x Means based on four replications; each conducted on a separate dish; values presented are arcsine transformed. y Germination least squares means for mean percent germination z Probability of a greater T value, under the null hypothesis that the mean equaled zero, based on percentage germination data. greenish-yellow fluorescent pigment when examined under the ultraviolet light. The standard identification system of ‘Appareils et Procedes d’identification’ (API) technique (API Systems, Biomerieux, SA, France) was used for biochemical tests. In in vitro experiment, isolates, Enterobacter sakazakii 8MR5, Pseudomonas sp. 4MKS8 and 10MKR4, Klebsiella oxytoca 10MKR7 were found to be promising rhizosphere bacteria that could stimulate the germination of S. hermonthica (Table 1). Pseudomonas sp. 4MKS8 stimu- lated the germination of seed of S. hermonthica by 3%. The percentage germination of S. hermonthica seed indu- ced by all the isolates (Table 1) was weak. Consequently, they were tagged low-level germination isolates. K. oxytoca 10MKR7 stimulated the germination of S. hermonthica seed to a limited degree, while the strigol analogue GR-24 induced effective germination in all replications with an average of 65% germination at a concentration of 1 ppm. Germination of S. hermonthica in water (control) was 0% in all tests. It was observed that germination percentages obtained with GR-24 solution vary from test to test despite the standard procedure used. Screen-house experiments The incidence of Striga plants over 9 week duration was recorded (Table 2) . Striga emergence was significantly stimulated over the control (water check). Throughout the entire experimental period, S. hermonthica emergence on the control pots was used as the standard for comparison. Percentage Striga emergence over the control pots showed that seed treatment with Pseudomonas sp. 10MKR4 gave the highest number of emerged Striga. This happened to be the least stimulant producer in the laboratory experiment followed by Pseudomonas sp. 4MKS8. Laboratory findings of Pseudomonas sp. 4MKS8 and E.sakazakii 8MR5 agreed with their screen-house findings. DISCUSSION This study demonstrates that there are some potential in certain rhizosphere bacteria to induce germination of S. hermonthica. This is in accordance with the observations of Babalola and Berner (2004) on length of the after- ripening period for S. hermonthica. The fact that the seeds were germinable, indicated that the synthetic strigol, GR- 24, induced effective germination of S. hermonthica seeds. The low stimulation of S. hermonthica by the bacterial isolates may be explained by the diffusion rates of the stimulant produced by the isolates. However, from the data, it was clear that GR - 24 is more effective than all the bacterial isolates in S. hermonthica seed germination stimulatory activity. That notwithstanding, the need for indigenous microbes in the integrated control method can not be overemphasized. The isolates tested could be low stimulant producers, such that, even statistically only stimulant from Pseudomonas sp. 4MKS8 is significant in the laboratory trial, but it is also possible that the seeds of the strain of S. hermonthica BD 97 do not respond to the isolates. Germination stimulant which are specific for S. hermon- thica strains have been reported by King and Zummo (1977) and Bebawi (1981). The isolates so far obtained were identified as low stimulant producers (Babalola et 4 Table 2. Stimulation of S. hermonthica as influenced by different rhizosphere bacteria applied by pelleted treatment on susceptible host plant sorghum (var. Mokwa Local). Selected bacterial isolate used Average number of Highest Striga count Highest Striga count as % in seed treatment emerged Striga per pot x per pot of count in water check Uninoculated control 8.2 14.1 100.00 Pseudomonas 10MKR4 15.0 30.3 214.89 K. oxytoca 10MKR7 13.9 25.5 180.85 Pseudomonas 4MKS8 13.4 28.0 198.58 E. sakazakii 8MR5 12.6 27.4 194.33 LSD (0.05) 3.68 CV (%) 36.3 x The soil was infested with 0.05 g Striga (about 3,000 germinable seeds out of about 10,000 total number of seeds) seed pot - 1 before sowing of sorghum seeds. et al., 2006). When stimulant activity was tested, in both laboratory and pot experiments, the percentage germina- tion effected by the stimulatory action of the bacteria was much lower than could be recommended for S. hermon- thica control without any other control method. The pur- pose of this development was to determine the bacterial stimulants for S. hermonthica, to release germination stimulants into the rhizosphere, and thereby further indu- cing the germination of S. hermonthica and consequently depleting the Striga seed bank when applied during trap cropping. These organisms that stimulate S. hermonthica seed germination may probably explain Striga decline in those sites for which there is no plausible explanation. ACKNOWLEDGEMENT Babalola O. O. had the visiting fellowship of the Interna- tional Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. REFERENCES Abbasher AA, Hess DE, Sauerborn J, Kroschel J (1996). Biological control of Striga. Paper presented at the ICRISAT sector review for Striga control in sorghum and pearl millet, 27-28 May 1996, ICRISAT- Bamako Mali. API. 20E (1988). Identification system for Enterobacteriaceae and other Gram-negative rods. Instruction Manual, Version D. No. 2010. Babalola OO (2002). Interactions between Striga hermonthica (Del.) Benth. and fluorescent rhizosphere bacteria Of Zea mays, L. and Sorghum bicolor L. Moench for Striga suicidal germination In Vigna unguiculata. PhD Thesis, University of Ibadan, Ibadan, 2002. Babalola OO, Berner DK (2004). Observations on preconditioning duration of Striga hermonthica on maize and sorghum yield attributes. Science Focus 8:79-82. Babalola OO, Osir EO, Sanni AI (2002). Characterization of potential ethylene-producing rhizosphere bacteria of Striga-infested maize and sorghum. Afr. J. Biotechnol. 1(2): 67-69. Babalola OO, Sanni AI, Odhiambo GD (2004). Isolation of rhizobacteria associated with maize and assessment of their potential for use in Striga hermonthica (Del.) Benth. suicidal germination. J. Trop. Microbiol. 3 (1):64 – 70. Babalola OO, Sanni AI, Odhiambo GD, Baldwyn T (2006). Plant growth- promoting rhizobacteria do not pose any deleterious effect on cowpea and detectable amounts of ethylene are produced. World J. Microbiol. Biotechnol. DOI: 10.1007/s11274-006-9290-6. Bebawi FF (1981). Intraspecific physiological variants of Striga hermonthica. Exp. Agric. 17:419–423. Berner DK, Kling JG, Singh BB (1995) Striga research and control a perspective from Africa. Plant Dis. 79:652-660. Eplee RE, Norris RS (1987). Field research techniques. In: Musselman LJ (ed) Parasitic weeds in Agriculture. Volume 1 Striga CRC Press, Inc., Boca Raton, Florida, pp. 271–280. King SB, Zummo N (1977). Physiological specialization in Striga hermonthica in West Africa. Plant Disease Reporter 61:770–773. Klyuchnikov AA, Kozhevin PA (1991). Dynamics of Pseudomonas fluorescens and Azospirillum-brasilense populations during the formation of the vesicular-arbuscular mycorrhiza. Microbiol. 59:449– 452. Mawdsley JL, Burns RG (1994). Inoculation of plants with a Flavobacterium species results in altered rhizosphere enzyme activities. Soil Biol. Biochem. 26:871–882. SAS/STAT User’s Guide (1989). volume 1, Acelus-freq SAS Institute Incorporation. SAS Users guide: statistics, version 6 Fourth Edition, pp. 1–890. Suslow TV, Schroth MN (1981). Bacterial culture preservation in frozen and dry-film methylcellulose. Appl. Environ. Microbiol. 42:872–877. Vancura V (1980). Fluorescent pseudomonads in the rhizosphere of plants and their relation to root exudates. Soil Sci. 18:185–190. Vancura V, Kunc F (1987). Interrelationships between micro-organisms and plants in soil. Proceedings International Symposium, Czechoslovakia. June 22-27. Czechoslovakia Academy of Science, Prague. Elsevier Tokyo, p. 492.