Br Effects of Plant Growth Promoting Rhizobacteria (pgpr) on Rooting and Root Growth of Kiwifruit (actinidia Deliciosa) Stem Cuttings

The effects of plant growth promoting rhizobacteria (PGPR) on the rooting and root growth of semi-hardwood and hardwood kiwifruit stem cuttings were investigated. The PGPR used were Bacillus RC23, Paenibacillus polymyxa RC05, Bacillus subtilis OSU142, Bacillus RC03, Comamonas acidovorans RC41, Bacillus megaterium RC01 and Bacillus simplex RC19. All the bacteria showed indole-3-acetic acid (IAA) producing capacity. Among the PGPR used, the highest rooting ratios were obtained at 47.50% for semi-hardwood stem cuttings from Bacillus RC03 and Bacillus simplex RC19 treatments and 42.50% for hardwood stem cuttings from Bacillus RC03. As well, Comamonas acidovorans RC41 inoculations indicated higher value than control treatments. The results suggest that these PGPR can be used in organic nursery material production and point to the feasibility of synthetic auxin (IBA) replacement by organic management based on PGPR.


INTRODUCTION
Kiwifruit are utilized almost exclusively as fresh fruits, with the exception of a small proportion juiced and mixed with other juices and used in wine coolers.The plant also has an ornamental value (1).
Kiwifruit was first introduced to Turkey in the 1980s.In the last few years significant progress has been achieved.Several experiments have been conducted in different agro-ecological areas; mainly in the Black Sea, Marmara, Aegean and Mediterranean regions, and considerable information has been collected on plantclimate and plant-soil relations and the area for kiwifruit cultivation in Turkey is expanding (2).Interest in self-rooting kiwifruit plants has increased, especially in some regions of Turkey where winterdamaged trunks can be replaced more rapidly from adventitious shoots.The demand for these plants has led nursery operators to look for effective means of propagating large number of plants rapidly.More recently, organic kiwifruit production has also gained importance in Turkey and there is increased interest among farmers in nursery materials for organically producued kiwifruit.
Kiwifruit may be propagated by various methods, such as grafting seedlings, stem cuttings, root cuttings and tissue culture (3).The most rapid and inexpensive production method of kiwifruit would be of considerable commercial value.Growing cuttings on their own roots could achieve this purpose by eliminating the need for producing rootstocks.Previous research has shown that kiwifruit stem cuttings are characterized by a variable rooting ability (4,5,6).Bench heating, mist, temperature control, growth substance treatments are always required to obtain satisfactory propagation in kiwifruit stem cuttings, making it one of the most difficult species to root (7,8).In addition, the type of cutting and the collecting time have been reported to strongly influence rooting of kiwifruit cuttings (9,10).
PGPR (plant growth promoting bacteria) have gained world wide importance and acceptance.The mechanisms involved in plant growth promotion by PGPR involve direct and indirect effects.Direct effects occur when PGPR produces substances such as phytohormones.These microorganisms are the potential tools for sustainable agriculture and the trend for future (11,12,13,14).Recent studies confirm that the treatments of seeds or cuttings with non-pathogen bacteria, such as Agrobacterium, Bacillus, Streptomyces, Pseudomonas, Alcaligenes etc. induced root formation in some plants because of natural auxin production of bacteria (14,15,16,17,18).Although, the mechanisms are not completely understood, root induction by PGPR is the accepted result of phytohormones, such as auxin production, inhibition of ethylene synthesis and mineralization of nutrients by PGPR (18,19).Considering the numerous interactions between the different hormonal signaling pathways in plants, it is difficult to assess which of these pathways is the primary target of PGPR.More likely, PGPR alters several, hormonal pathways.This could account for the different morphological changes observed, for example, lateral root elongation and root hair development.One of the more characteristic effects of PGPR is an increased elongation rate, and perhaps initiation rate, of lateral roots resulting in more branched root system architecture (20,21).
Environmentally friendly applications in agriculture have gained more importance, in particular in horticulture and nursery production.The use of PGPR for nursery material multiplication may be important for obtaining organic nursery material because the use of all formulations of synthetic plant growth regulators, such as indole-3-butyric acid (IBA), is prohibited in organic agriculture throughout the world.
The aim of this study was to determine the effects of PGPR strains, Bacillus RC23, Paenibacillus polymyxa RC05, Bacillus subtilis OSU 142, Bacillus RC03, Comamonas acidovorans RC41, Bacillus megaterium RC01 and Bacillus simplex RC19 on rooting and root growth in kiwifruit cv.Hayward semi-hardwood and hardwood stem cuttings.In vitro production of indole-3-acetic acid (IAA) by PGPR was also evaluated.

Bacterial strains, isolation and identification of bacteria
The PGPR used in this study are given in Table 1.The seven non-pathogenic bacteria, Bacillus RC23, Paenibacillus polymyxa RC05, Bacillus subtilis OSU 142, Bacillus RC03, Comamonas acidovorans RC41, Bacillus megaterium RC01 and Bacillus simplex RC19 were initially isolated in late summer from the rhizosphere of wild raspberry, wheat and tomato plants in northeastern Turkey, except for Bacillus subtilis OSU 142 from Ohio, USA.The isolation of bacteria was carried out according to Ramos Solano et al. (22).The bacteria were identified based on their whole-cell fatty acid methyl ester (FAMEs) analysis (23) using the MIDI system (Sherlock Microbial Identification System version 4.5, MIDI, Inc., Newark, DE).Bacteria were grown on nutrient agar (NA) for routine use, and maintained in nutrient broth (NB) with 15% glycerol at _ 80ºC for long-term storage.For each experiment, a single colony was transferred to 500 ml flasks containing NB, and grown aerobically in flasks on a rotating shaker (150 rpm) for 48 h at 27ºC (Merck KGaA, Germany).The bacterial suspension was then diluted in sterile distilled water to a final concentration of 10 9 CFU ml -1 , and the resulting suspensions used to treat kiwifruit cuttings.

Quantification of IAA production of bacteria
The bacteria were also tested for indole-3acetic acid (IAA) production, using the method of Bent et al. (24).The flasks were incubated for 18 h at 27°C with 100 rpm rotary shaking.Following this, 125 ml flasks containing 40 mL half-strength TSB, supplemented with 0, 0.1, and 25 mg tryptophan ml -1 were each inoculated with 1ml of each strain.After incubation for 48, 72, and 168 h, the density of each culture was measured spectrophotometrically at 600 nm, and then the bacterial cells were removed from the culture medium by centrifugation.The level of indoles present in the culture fluid was estimated colorimetrically.The concentration of IAA in the bacterial eluates was measured by using Salkowski's reagent (50mL 35% HClO 4 + 1mL FeCl 3 ).Each reaction mixture was centrifuged.The absorbance at 530 nm in a Shimadzu Spectrophotometer UV-1208 was measured.Bacterial cells were separated from the supernatant by centrifugation at 10,000 rpm for 30 min.The concentration of IAA in each culture medium was determined by comparison with a standard curve.The IAA produced by each strain was measured in triplicate.Besides, after 48, 72, and 168 h of growth, samples were taken for determination of IAA by thin-layer chromatography (TLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis.Separation of indole in ethyl-acetate fraction was carried out in chloroform-ethyl acetate-formic acid.

Plant materials
Semi hardwood and hardwood stem cuttings of kiwifruit cv.Hayward were prepared from middle parts of vigorous shoots of 14-yr-old plants during June and January in both 2006 and 2007.The cuttings had three buds and were 20 cm in height and 1 cm in diameter.Bacterial treatments were performed by dipping the cuttings into the bacterial suspension prepared in sterile water at the concentration of 10 9 cfu ml -1 for 30 min.Cuttings in the control group were treated with sterile water.For IBA treatments, the basal portion of cuttings was dipped in an aqueous solution of either 2000 or 4000 ppm IBA (50% ethanol) for 5 min, and allowed to air dry.Following treatments, cuttings were placed in trays filled with perlite media to a depth of 10 cm under mist (15 s/6 min) in a greenhouse maintained at 21 ± 2 0 C. The data were obtained after 3 months.The evaluated parameters determined by Orhan et al. (25) were rooting percentage, the number of main roots per cutting, the highest root length (cm), average root diameter (mm), root dry weight per cutting (mg) and root quality (1-5 scale).

Data Analysis
The experimental design used was a randomized complete block with 4 replications.Each replication contained 10 cuttings spaced 50 mm apart.Data were subjected to analysis of variance using ANOVA and means were separated by Duncan's multiple range tests (p<0.05).There were no statistical differences between years, therefore the data were pooled.(OD 600 unit) -1 ), while the lowest IAA production was detected from Bacillus RC23 (20.4 μgml -1 (OD 600 unit) -1 ) (Table 2).Many micro organisms that interact with plants can synthesize hormones similar to those produced by the plant as growth regulator, such as auxins, gibberellins and cytokines (26).Among them, auxin is one of the most well-known phytohormones because of its important role in the initial processes of lateral and adventitious root formation (27) and root elongation (28).
Our results showed that PGPR produce auxin themselves and may also affect auxin synthesis of cuttings (29).IAA is the most commonly produced auxin in nature, synthesized mainly through tryptophan dependent pathways.The endogenous level of IAA in the plant is also important for successful rooting.Furthermore, other indolic compounds, such as indole-pyruvic, indole-acetamide and indole-carboxylicacid, can be involved in root formation (30).

Rooting and root growth of stem cuttings
The data obtained on the number of main roots per cutting, the highest root length, average root diameter, root dry weight, root quality and rooting percentage for each treatment in kiwifruit semi-hardwood and hardwood stem cuttings are shown in Table 3 and Table 4. Great variation on all parameters was observed among the PGPR tested in both semi-hardwood and hardwood stem cuttings types (p<0.05)(Table 3 and 4).
In semi-hardwood cuttings, in terms of induction of roots, the different treatments exhibited varying degrees of response.The PGPR treated semi-hardwood stem cuttings of kiwifruit generally had significantly higher numbers of main roots, root length, root diameter, root dry weight, root quality and rooting percentage than water-treated control stem cuttings.However, in general these parameters were lower than 2000 ppm and 4000 ppm IBA (indole-3-butyric acid) treatments.
The best root dry weight (mg) was observed in the 4000 ppm IBA treatment as 22.33 mg, while the lowest in control cutting was 2.56 cm (Table 3).
The root quality was the highest in the 4000 ppm IBA treatment (3.33), followed by the 2000 ppm IBA treatment (3.02) and Paenibacillus polymyxa RC05 (2.82).The root quality in control cuttings were the lowest among the treatments (1.63) (Table 3).
The differences in rooting with different treatments were significant, with the highest rooting percentage observed in 4000 ppm IBA, followed by 2000 ppm IBA (57.50%), and equally 47.50% in Bacillus RC03 and Bacillus simplex RC19, respectively.The control treatment gave the lowest rooting percentage (12.50%)(Table 3).
Increases in rooting percentage and root growth of cuttings varied according to PGPR tested.All PGPR increased root growth and rooting percentage over the control.Among PGPR treatments, Bacillus RC03 were found more effective on the number of main root (3.53) and rooting (42.50%),Bacillus simplex RC19 on the highest root length (7.27 cm), Bacillus RC23 on root diameter (1.22 mm), Paenibacillus polymyxa RC05 on root dry weight (19.32 mg) and root quality (2.44) and Comamonas acidovorans RC41 on rooting (42.50%), respectively.Control cuttings gave 0.00% rooting percentage (Table 4).
These results show that the PGPR treatment is useful for root induction in kiwifruit stem cuttings.Auxin production by bacteria is one of the explanation of root induction in kiwifruit stem cuttings.Another explanation could be that the cuttings can produce auxin themselves after PGPR inoculation.Unfortunately we did not analyze auxin levels in cuttings during the experiment.The PGPR produced higher percentages of root growth and better quality in terms of root length, diameter, root dry weight etc (Table 3 and Table 4).PGPR is able to exert a beneficial effect on plant growth such as increases in root growth and weight (31).It is evident that the treatment of PGPR on cuttings of different plant species showed genotype dependent rooting and increased root growth (32,33).Patten and Glick (34)  In conclusion, this study demonstrated that the PGPR belonging to genus Bacillus, Paenibacillus and Comamonas has potential to promote root formation in kiwifruit cuttings in mass clonal propagation.It seems that the stimulation of rooting and root growth by PGPR can be correlated to production of indole-3-acetic acid by the bacteria.In our study in general, the PGPR has higher IAA producing capacity, which also resulted in higher rooting percentages in cuttings.Among PGPR used, as mentioned before, Bacillus RC03, Bacillus simplex RC19 and Comamonas acidovorans RC41 were found to be produce more IAA and these bacteria are also more effective on rooting in kiwifruit stem cuttings, maximizing yield of rooted clonal cuttings in nurseries.As is well-known, the use of chemicals in plant propagation can produce environmental problems and may increase the cost of propagation in nurseries.Therefore, these results can also be important for the use of these PGPR to multiply organic nursery materials.In fact, several commercial PGPR products are already being used in organic agriculture practice.A further topic to analyze is whether the cuttings produce auxin themselves after PGPR treatments.

TABLE 1
The bacterial strains, codes and sources

TABLE 2
The production of IAA by PGPR in the presence of various concentrations of tryptophan * Values in the same column with different lower-case letters in same clone are significantly different at p<0.05.Average ± standard error from three separate experiments.Data were means of three replicates IAA production in average 48, 72, and 168 h pure cultures.

TABLE 3
The effect of bacteria on rooting and root growth of semi-hardwood cuttings of kiwifruit cv.Hayward (Average of 2006 and 2007 years) * Values in the same column with different lower-case letters are significantly different at p<0.05.

TABLE 4
The effect of bacteria on rooting and root growth of hardwood cuttings of kiwifruit cv.Hayward (Average of 2006 and 2007 years) * Values in the same column with different lower-case letters in same clone are significantly different at p<0.05.