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Improvement of grafting efficiency in hard grafting grape Berlandieri hybrid rootstocks by plant growth-promoting rhizobacteria (PGPR)
Scientia Horticulturae 164 (2013) 24–29
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Improvement of grafting efficiency in hard grafting grape Berlandieri hybrid rootstocks by plant growth-promoting rhizobacteria (PGPR) Ali Sabir ∗ University of Selcuk, Faculty of Agriculture, Department of Horticulture, 42075 Konya, Turkey
a r t i c l e
i n f o
Article history: Received 30 May 2013 Received in revised form 22 August 2013 Accepted 23 August 2013 Keywords: Green grafting Callus formation Graft survival PGPR
a b s t r a c t Certain grapevine rootstocks in Vitis berlandieri hybrids (such as 41 B, one of the most important rootstocks used in worldwide viticulture) have great potential to use owing to its great resistance to calcareous soils and phylloxera. But their use is limited due to the difficulties in propagation and grafting. PGPR is reported to stimulate division and enlargement of plant cells that might result in better fusion at graft union point. This investigation was thus conducted to reveal the effects of plant growth promoting rhizobacteria (Pseudomonas putida strain BA-8, Bacillus simplex strain T7) on callus formation, graft success, vegetative development and subsequent bud fruitfullness of Alphonse Lavallée grape cultivar. The green softwood shoots of Alphonse Lavallée (AL) grape were grafted on the rootstocks 41 B or 1103 P by modified cleft green-grafting technique in controlled glasshouse. Before grafting, the single scion nodes were divided into four treatment groups. Three of them were separately dipped into bacterial suspension (109 CFU ml−1 ) of either of two strains or their combination for an hour. One of the treatment groups was dipped into the tap water (as control). Each treatment has contained thirty grafts, divided into three replicates. In both graft combinations, combined application of BA-8 and T7 strains resulted in 100% callusing compared with the uninoculated control where the percentages were 93.3 and 90.0% for AL/41 B and AL/1103 P combinations, respectively. Individual or combined applications of the strains also had remarkably positive influences on full callusing rates. The highest shoot length values were obtained from combined application (113.5 cm) and BA-8 strains (100.9 cm) in AL/41 B and AL/1103 P graft combinations, respectively. Inoculation of the graft scion parts with BA-8 strain resulted in the highest shoot lignification lengths in both AL/41 B and AL/1103 P combinations with the values of 75.4 and 75.6 cm, respectively while uninoculated grafts of the representing combinations were as low as 59.0 and 55.3 cm. In AL/41B, the highest (100%) graft survival rate in nursery was recorded in plants treated with bacterial combination. Combined application as well as BA-8 performed better in AL/1103P. The findings imply that PGPR can be employed in ecological and sustainable production of nursery material and point to the feasibility of commercially synthetic auxin replacement by PGPR-based ecological treatment. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In modern cropping systems, grafting is commonly used to impart pathogen resistance, manipulate grapevine physiology, confer disease or pest resistance, and tolerate certain soil types (Gambetta et al., 2009). Grape rootstocks have been bred to provide resistance to improper soil condition, diseases such as phylloxera (Daktulosphaira vitifoliae Fitch) or other pathological problems. The practice of grafting onto disease-resistant stocks now extends to a variety of horticultural plants. However, mistakes in any stages of grafting can result in serious problems difficult to compensate. Many factors may affect the success of grafting such as time of
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grafting, cold treatment of the cuttings and hormonal application. Anatomically, the cambium of a stock and scion must be in close contact to form a union. During the grafting process, undifferentiated callus cells are formed, which will differentiate into specialized cells that form a new xylem (water and nutrient pathway) and phloem (sugar pathway) within the graft union. Several different grafting techniques can be performed to generate complete grapevines. Among them, green grafting offers growers to propagate the desired cultivars during the growing period after the time for grafting with dormant scions has ended. In this technique, succulent green current-season growth is used as scions that are grafted on the new growth of cultivar rootstock. Furthermore, green grafting can be performed early as shoots are large and firm enough to handle. The method has been successfully adopted in many countries, where grape cultivation is common (Kaserer et al., 2003). Green grafting technique has many
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other advantages such as easiness for application, relatively more efficient graft-scion affinity to overcome the graft incompatibility sometimes experienced between distantly related Vitis species (Bouquet and Hevin, 1978; Sabir and Kara, 2004). This technique is currently being used for rapid and routine indexing of grapevine virus diseases (leafroll, fleck, corky bark, vein mosaic, vein necrosis) and more recently as a means of evaluating virus presence in certification programs (Walker and Meredith, 1990; Walter et al., 1990; Pathirana and McKenzie, 2005). Currently, application of concrete grafting types has gained importance for obtaining successful result, although little data exist regarding the advantages and disadvantages of different green-grafting methods (Sabir, 2011). Some grapevine rootstocks such as Vitis berlandieri have great potential to be used as grape rootstock owing to its resistance to calcareous soils and phylloxera, but their use is limited due to the difficulties in propagation and grafting. This is a common problem in rooting or grafting of 41 B rootstock although it is one of the most important rootstocks used around the world. For a successful propagation, increasing callus formation, grafting union, and grafting success are very important, and PGPR can have effects on success rate (Maheshwari, 2011). The effectiveness of grafting zone inoculation with Pseudomonas BA-8 (cytokinin producer) and Bacillus BA16 and OSU-142 (IAA producers) to improve successing rate, callusing rate, callusing degree, and full callusing rate on four different scion-rootstock combinations were studied by Kose et al. (2005). All the strains significantly increased success rate of grafting in varying degrees among the grafting combinations compared to the noninoculated control. Some of such PGPR classified as endophytes are able to transcend the endodermis barrier, crossing from the root cortex to the vascular system, and subsequently thrive as endophytes in stem, leaves, tubers, and other organs (Compant et al., 2005). The extent of endophytic colonization of host plant organs and tissues reflects the ability of bacteria to selectively adapt to these specific ecological niches (Gray and Smith, 2005; Esitken et al., 2006). As a result, intimate associations between bacteria and host plants can be formed without harming the plant (Lodewyckx et al., 2002). PGPR colonize the plant rhizosphere and positively affect their growth and responses to stress (Compant et al., 2008; Yang et al., 2009). In general, PGPR treatments were given to roots, but recently, sprayed to aerial part of plants (Esitken, 2011). To our knowledge, surprisingly, very little is known about the effects of PGPR on graft success in grapevine as well as other perennials, except for a few studies (Kose et al., 2005). Therefore, this investigation was conducted to evaluate the effects of plant growth promoting rhizobacteria (Bacillus simplex T7 and Pseudomonas putida strain BA-8) on callus formation, graft success and early development of scion shoots of Alphonse Lavallée grape cultivar on two different rootstocks 41 B (very difficult to root with a low graft success) and 1103 P (moderate rooting and grafting success) using a relatively new and high-success grafting type.
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on the rootstocks by modified cleft green-grafting technique. The green softwood shoots chosen for grafting had an equal diameter to the rootstocks (5–8 mm), and were at 3–6 nodes below the active bud (Walter et al., 1990). Modified cleft grafting was applied as described previously by Sabir (2011). A slit was made down the center of the scion by a razor blade and a long tapered wedge was made on the opposing piece (rootstock). For all grafts, the shoots of both rootstock and the scion had the same diameter at the point of graft contact so that the cambium layers of the union matched (Martelli et al., 1993). The assembled grafts were wrapped with commercial plastic bandage tape. Each rootstock plant had one graft scion and the remaining shoots on grafted plants were discarded. 2.2. Bacterial inoculum and plant bacterization The grafted plants were randomly divided into 4 treatment groups. Three groups of the prepared scion parts were separately immersed into individual or combined bacterial solutions of cytokinin synthesizing Pseudomonas putida strain BA-8 and auxin synthesizing Bacillus simplex strain T7 (109 CFU ml−1 ) in 2 l containers and incubated at room temperature for an hour. One of the treatment groups was dipped into the tap water and used as a control. After treatments, grafting was performed immediately by using modified cleft method. For each bacterial treated and control groups, two different combinations of rootstock-scion including Alphonse Lavallée/41 B and Alphonse Lavallée/1103 P were used. 2.3. Growth condition The graft union was healed under condition of moderate light, fairly high humidity (between 60 and 80%) and good air circulation. The grafted plants were kept under tulle curtain having 50% transmission, with mean daily temperature in the glasshouse ranged between 21 and 30 ◦ C (according to the temperature and humidity data logger) during the experiment over 3 weeks. Then, the curtain was removed to allow the scion shoot to harden properly. Night and day temperatures during the vegetative development period were 20 ± 3 and 28 ± 4 ◦ C respectively. To achieve a concrete irrigation, two soil moisture sensors (Irrometer company, Riverside, CA) were installed 30 cm depths to continuously monitor changes in soil–water content (http://www.irrometer.com). The irrometer readings were taken every day to maintain the soil moisture at the field capacity level according to the manufacturer’s guideline. To ensure the uniformity of irrigation, the water was transported directly into the pots by micro irrigation systems consisted of individual spaghetti tubes. Lateral shoots emerging from the active summer buds of plants were removed daily, allowing only the main shoots to elongate in order to ensure valid comparison of development. The shoots were tied with thread to wires established 2.5 m above the pots to let the plants grow on a fence in an upright position, thus ensuring equal benefit from the light. 2.4. Plant analyses
2. Materials and methods 2.1. Plant material and graft operation The study was established in an experimental glasshouse of Selcuk University, Agriculture Faculty, Horticulture Department, Konya, Turkey. One year old own rooted 41 B (Vinifera x Berlandieri) and 1103 P (Berlandieri x Rupestris) vines in equal vegetative growth were individually cultivated in 8 l plastic pots containing uniform mixture of peat (1.034% N, 0.94% P2 O5 , 0.64% K2 O, pH 5.88, Klassman® ) and inert perlite (0–3 mm in diameter) in equal volume. After a two-month vegetative development stage in early spring of 2012, grapevine cultivars Alphonse Lavallée was grafted
For the analyses of callusing response of the grafts to bacterization, graft unions were visually evaluated 4 weeks after grafting operation for callusing rate, full callusing rate and callusing degree as described previously by Hartman et al. (1990), Baydar and Ece (2005) and Sabir (2011). Callusing rate was determined as percentage by using callus formation around the graft cut surfaces. Full callusing rate was the percentage of grafts of which graft cut surfaces callused at 100%. Callus degree was determined by using 1–4 scale; 1: 1–25% callusing, 2: 26–50% callusing, 3: 51–75% callusing and 4: 76–100% callusing at graft union point. In order to investigate the growth promoting effect of bacterization, measurements on shoot length (scion shoot was measured with a sensitivity of 1 mm),
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A. Sabir / Scientia Horticulturae 164 (2013) 24–29
Fig. 1. Early development course of AL/41 B with respect to bacteria applications. Error bar stands for the standard deviation of the means.
Fig. 2. Early development course of AL/1103 P with respect to bacteria applications. Error bar stands for the standard deviation of the means.
shoot lignification length (the length of the scion shoot, where complete lignification occurred) and shoot diameter (measured by digital compass at a point 1 cm above the second node). Early shoot development was also investigated by measurements performed with 2–4 days intervals. Besides, fruitfulness response of bacterized grafts was investigated at the beginning of the growth season of the following year (2013). For this, each plant in glasshouse was pruned back at the end of the winter season after experimental growth season leaving two winter buds per plant (considering the genetic bud fruitfulness potential of Alphonse Lavallée cultivar). After bud sprout and subsequent two-month-shoot development, clusters born on the one-year-old grafts were calculated.
apparent in the grafts. The response of different graft combinations to bacterial inoculums differed with respect to early development of scion shoots. Individual or combined applications of the bacteria strains significantly (P < 0.05) increased the scion shoot length, shoot lignification length and shoot diameter of all the graft combinations on both the rootstocks (Table 2). The highest shoot length values were obtained from combined application (113.5 cm) and BA-8 strains (100.9 cm) in AL/41 B and AL/1103 P graft combinations, respectively. Inoculation of the graft scion parts with BA-8 strain resulted in the highest shoot lignification lengths among the AL/41 B and AL/1103 P combinations with the values of 75.4 and 75.6 cm, while uninoculated grafts of the representing combinations were as low as 59.0 and 55.3 cm, respectively. Shoot diameter response of the grafts were also in accordance with the results obtained from shoot lignification length. The grafts inoculated with the stains of BA-8 had the highest shoot diameter values in both graft combinations. As can be seen in Table 3, all bacterial inoculations increased the leaf (node) number across the grafts. In particular, the most effective application was combined strains. To illustrate, the highest values of leaf number per plant were 25.3 and 26.7 for AL/41 B and AL/1103 P combinations, while uninoculated controls had 20.0 and 21.5 leaves per plant, respectively. Individual treatments of bacteria strains had little or no effect on leaf area of the grapevines. However, combined applications significantly (P < 0.05) increased the leaf area in AL/41 B and AL/1103 P grafts with their values 95.4 and 91.7 cm2 while the controls of the same grafts had 86.4 and 82.7 cm2 , respectively. Pruning residue was also a good indicator of the response of grapevine grafts to inoculation with bacteria strains. Inoculation induced an enhancement of shoot pruning residue weight as well as the number of nodes. As presented in Fig. 3, in AL/41B, the highest (100%) graft survival rate was recorded in plants treated with bacterial combination. Also, combined application as well as BA-8 was better in AL/1103P. Bacteria strains clearly enhanced the graft success, although their effects were cultivar-dependent.
2.5. Statistical analysis The study design was a complete randomized block with three replicates. Each replicate consisted of 10 grafted plants. Data for each rootstock were separately evaluated by analysis of variance (ANOVA) and treatment means were separated by Least Significant Differences (LSD) test at P < 0.05. Data were analyzed using SPSS program version 13.0 (SPSS Inc., Chicago, IL). 3. Results 3.1. Callusing response of the grafts to bacterization Inoculation of PGPR strains alone or in combination significantly (P < 0.05) increased callusing rate, full callusing rate and callusing degree accross the graft combinations, except for callusing rate in Alphonse Lavallée (AL)/41 B (Table 1). In both graft combinations, combined application of Pseudomonas BA-8 and Bacillus simplex T7 strains resulted in 100% callusing compared with the uninoculated control where the percentages were 93.3 and 90.0% for AL/41 B and AL/1103 P, respectively. Similarly, bacteria strains significantly (P < 0.05) improved full callusing rates in grafts, with the highest rates obtained from combined application across the grafts. As for the callusing degree around the graft union point, individual or combined applications of the strains had also remarkably positive influences with the highest degrees being promoted by combined application in AL/41 B (3.84) and BA-8 strains in AL/1103 P (3.95). 3.2. Growth promoting effect of bacterization In the scope of comparing early shoot developments among the experimental groups, shoot length measurement performed with 2–4 days intervals in early development stage revealed that bacteria applications had significantly (P < 0.05) positive effect on AL/41 B grafts (Fig. 1) and slight effect on AL/1103 P combination (Fig. 2). Positive effects of combined application or BA-8 alone were more
Fig. 3. Survival rate responses of 41 B and 1103 P grafts to bacterization. Each column represents the mean of triplicate determinations with ten grafts for replicate. Error bar stands for the standard deviation of that mean.
A. Sabir / Scientia Horticulturae 164 (2013) 24–29
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Table 1 Effects of bacterial strains on callus formation of different graft combinations. Scion/rootstock
Inoculant
Callusing rate (%)
Full callusing rate (%)
Callusing degree (1–4)
AL/41B
Control T7 BA-8 T7+BA-8
93.3 ± 5.77 100 ± 0.00 96.7 ± 5.77 100 ± 0.00
56.7 ± 5.77 c 73.3 ± 5.77 b 70.0 ± 0.00 bc 86.7 ± 5.77 a
3.51 ± 0.08 c 3.74 ± 0.06 ab 3.67 ± 0.11 bc 3.84 ± 0.04 a
ns
10.8
0.14
90.0 ± 0.00 b 96.7 ± 5.77 a 100 ± 0.00 a 100 ± 0.00 a
60.0 ± 0.00 b 76.7 ± 5.77 a 83.3 ± 5.77 a 90.0 ± 0.00 a
3.38 ± 0.20 b 3.90 ± 0.09 a 3.95 ± 0.08 a 3.85 ± 0.14 a
5.52
7.65
0.25
LSD (%5) AL/1103P
Control T7 BA-8 T7+BA-8
LSD (%5)
Levels not connected by same letter are significantly different; AL, Alphonse Lavallée; and ns, not significant.
Table 2 Effects of bacterial strains on shoot developments of different graft combinations. Scion/rootstock
Inoculant
Length (cm)
Lignification length (cm)
Diameter (mm)
AL/41B
Control T7 BA-8 T7+BA-8
73.1 ± 2.47 d 82.9 ± 3.35 c 95.1 ± 2.71 b 113.5 ± 7.07 a
59.0 ± 5.51 b 57.9 ± 0.90 b 75.4 ± 2.25 a 75.2 ± 3.61 a
4.03 ± 0.10 c 4.17 ± 0.04 b 4.48 ± 0.06 a 4.37 ± 0.05 a
8.1
6.60
0.12
84.7 ± 4.73 b 96.3 ± 3.81 a 100.9 ± 2.83 a 100.3 ± 7.76 a
55.3 ± 3.77 b 71.9 ± 1.90 a 75.6 ± 4.00 a 74.8 ± 3.38 a
4.39 ± 0.03 c 4.34 ± 0.05 c 4.65 ± 0.03 b 4.72 ± 0.02 a
9.63
6.30
0.07
LSD (%5) AL/1103P
Control T7 BA-8 T7+BA-8
LSD (%5)
Levels not connected by same letter are significantly different; AL, Alphonse Lavallée; and ns, not significant.
Table 3 Effects of bacterial strains on leaf development and pruning residue of different graft combinations. Scion/rootstock
Inoculant
Leaf (node) number
Leaf area (cm2 )
Pruning residue (g)
AL/41B
Control T7 BA-8 T7+BA-8
20.0 ± 0.23 c 20.3 ± 0.25 c 23.4 ± 0.81 b 25.3 ± 0.78 a
86.4 ± 1.65 b 93.4 ± 1.76 a 85.9 ± 2.49 b 95.4 ± 2.64 a
7.53 ± 0.15 c 7.43 ± 0.12 c 9.89 ± 0.00 b 11.88 ± 0.43 a
1.10
4.09
0.45
21.5 ± 0.46 c 24.2 ± 0.20 b 25.6 ± 0.95 a 26.7 ± 0.46 a
82.7 ± 3.30 b 82.5 ± 1.68 b 84.3 ± 1.62 b 91.7 ± 2.25 a
9.87 ± 0.45 c 11.65 ± 0.55 b 13.47 ± 0.40 a 13.24 ± 0.21 a
1.10
4.05
0.78
LSD (%5) AL/1103P
Control T7 BA-8 T7+BA-8
LSD (%5)
Levels not connected by same letter are significantly different; (mean ± SD); AL, Alphonse Lavallée; and ns, not significant.
3.3. Fruitfulness response of bacterized grafts According to the findings obtained in the beginning of the following vegetation period, all strains significantly (P < 0.05) increased the number of cluster per plant (Fig. 4) across the grafts on both 41 B and 1103 P, compared with the uninoculated control, the maximum increase being promoted by BA-8 in 41 B and combined treatment in 1103 P. In AL/41 B grafts, BA-8 induced more than two fold increment in cluster number per young vines while a very similar inducement was obvious in combined treatment of AL/1103 P.
agricultural techniques and bio-fertilization are vital to alleviating environmental pollution. Over the past two decades, research on bacteria species has been steadily expanding as the strains of various bacteria ecologically enhance plant growth, disease
4. Discussion Intensive agriculture still relies on the use of chemical fertilizers to provide faster vegetative growth and high fruit yield although excessive use of chemical fertilizers causes problems not only in terms of financial cost but also in terms of the cost to the environment. Therefore, the development and application of sustainable
Fig. 4. Cluster development responses of 41 B and 1103 P grafts to bacterization. Each column represents the mean of triplicate determinations with ten grafts for replicate. Error bar stands for the standard deviation of that mean.
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resistance, and improve nutrient fixation or solution (Ait-Barka et al., 2006; Karakurt and Aslantas, 2010). Such findings have stimulated a growing interest for using PGPR isolates in viticulture for various purposes (Esitken, 2011). However might it very be surprising, literature data to investigate the effects of PGPR strains on graft success increment in Vitis species is insufficient although it has been very well-known that certain bacteria species produce auxin and/or auxin like plant growth promoting substances (Kose et al., 2005). In the present study, the bacteria strains Pseudomonas BA-8 (cytokinin producer) and Bacillus simplex T7 (auxin producer) stimulated the formation of callus tissue around the graft point. Combined application of the bacteria strain had more pronounced positive effect on callus formation parameters in AL/41 B while BA-8 was withstanding with its individual positive effect in AL/1103 P. This finding is in accordance with the findings of Aslantas et al. (2007) who observed higher IAA level produced by BA-8 among three different strains (Pseudomonas BA8, Bacillus BA16 and Bacillus OSU142) by HPLC detections. Also, previous studies verified that strains in the genus studied in the present study Pseudomonas and Bacillus were able to produce plant growth regulators particularly indole-3-acetic acid (IAA) (Lambrecht et al., 2000). According to the tissue analyze studies, an increasing number of IAA-producing plant growth-promoting rhizobacteria (PGPR) are detected inside the plant tissue in relation with the amount of bacteria (Rosenblueth & Martinez-Romero, 2006). In addition to this, Grichko and Glick (2001) set forth that Pseudomonas strains may have ACC deaminase enzyme which can cleave the plant ethylene precursor ACC and thereby lower the level of ethylene in plants. Adhesion between the rootstock and the scion is aided by a socalled tissue “callus”. Wound-repair xylem is generally the first differentiated tissue to bridge the graft union. Aside from whatever role IAA plays in promoting cell division (callus formation) during graft union formation (Yin et al., 2012), IAA and cytokinin plays an important role in xylem formation across the callus bridge between stock and scion. Further, cambial continuity after callusing between graft partners has also an essential role in graft survival rate (Sabir, 2011). From a practical perspective, however, exogenous application of auxin in grafting has not caught on, because the response of Vitis materials is small, if not, and variable (Sabir and Agaoglu, 2009). Therefore the bacterial source of IAA and cytokinin application to promote formation of new vascular tissue promises potential and sustainable advances. Previously Esitken (2011) stated that PGPR, with their various hormonal potentials, can be used for various purposes such as rooting of cutting, grafting union, fruit setting and thinning, lateral root formation, increasing tolerance against abiotic stress as well as growth, development, and biological control with root inoculation and/or spraying. The growth inducing effects of the bacteria strains revealed in the present study verifies the previous findings obtained by Esitken et al. (2003) in apricots and Aslantas et al. (2007) in apples where inducing effects of especially BA-8 strains on shoot growth were documented. Among the physiological mechanisms pertaining to vegetative growth inducement, according to the literature, the most important one might be related to bacterial production of the phytohormone IAA (Frankenberger and Arshad, 1995). Studies also indicated that the extent of positive effects of various bacterial strains on plant growth varied with the species or variety of host plant (Alam et al., 2003; Kose et al., 2005; Sabir et al., 2012), as seen in this study with different response of grafts. In this study, early shoot development of bacteria inoculated plants were also better than those of control ones. This outcome supports the results of a previous study that reveals the movement of vascular bacteria between scion and rootstocks (Gambetta et al., 2009). The researchers demonstrated that the bacteria moves through open xylem conduits of grapevine graft union and may carry out systemic
infection in vine tissues. Using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM), Piccolo et al. (2010) and Compant et al. (2011) also detected the distribution of various bacterial colonies in seeds, flowers, leaf veins, cells, hairs, intercellular spaces, and cut edges of leaf disks of five different grape cultivars. They further identified the bacteria strains such as Bacillus spp., Burkholderia spp., Pseudomonas spp., in many samples by 16S rRNA gene sequencing or microscopic investigations. Ait-Barka et al. (2006) demonstrated that PGPR Burkholderia phytofirmans strain PsJN increased grapevine growth and physiological activity even at low temperatures. The mentioned literature, including the present findings, suggests the potential role of bacteria as plant growth promoters and sources of plant growth manipulating substances. Apart from the success in graft and callus formation, survival rate in nursery and early growth in vineyard is essential for a profitable use of agricultural area (Sabir et al., 2012). In the current study, bacteria inoculation alone or in combination markedly helped the young plants to survive. Previously, using different hardwood grafting type and varieties, Kose et al. (2005) demonstrated that there were statistically important increases in the survival of grafts in relation with PGPR applications. Previous report set fort that the PGPR stick to plant cell within an hour after inoculation and migrate into the entire plant parts including leaves and inflorescences (Compant et al., 2005, 2008) and then an interaction between plant tissue and the bacteria is established. The sustained growth responses in both vegetative and generative aspects investigated in this study seem to be in accordance with the reports mentioned above. Bacterized graft shoots not only grew faster than nonbacterzed controls but also were sturdier, with a better developed thick shoots. This, combined with an enlarged leaf growth may have contributed to the stimulation of generative development, namely floral changes which occur during early shoot development stage in buds. Because, the plants having much more morphological development under the bacteria effects were also having significantly greater cluster numbers born in the following year of this study. Previous studies indicated that the high concentrations of cytokinin and IAA levels in leaves and nodes of perennial plants during the induction and initiation of floral periods, may physiologically promote flower formation (Koshita et al., 1999; Ulger et al., 2004). These literature and the present results verify that the bacterial inoculation resulted in better floral development with their growth inducing effects emerging from consolidated hormone levels in newly grafted plants.
5. Conclusion Overall investigations in the present study indicated that individual and/or combined applications of the strains of BA-8 and T7 have significantly positive influences on graft callusing, scion shoot growth, cane hardening, and nursery survival rate as well as fruitfulness of the grapes in following year. Combined application of bacteria strains displayed more pronounced effects on most of the investigated criteria. Callus promoting potential of the strains clarified their ability to synthesize plant growth regulators. The findings of current study is anticipated to shed light into the application and sustainable use of bacterial bioinoculants in enhancing graft success, survival rate, nursery and young vineyard development as well as fruitfulness and cold hardening of grapevines. Considering the large lose in acclimatization stage of intact- and nursery-grown grapevines, especially when 41 B rootstock is used, the employment of PGPR promises positive effects on enhancement of graft success. Therefore, further studies may be useful to sucrutinize the cultivar-specific activities of PGPR with
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physiological and anatomic mechanisms underlying the investigated growth. Most of the viticulture area on the world today face with serious problems emerging from biotic or abiotic stress factors such as phylloxera, nematodes, water shortage, winter cold, excessive limestone, etc. Therefore, practical application studies on the sustainable use of such PGPRs under stress condition will be our next step toward a better benefiting the role of microorganisms. The results finally imply that such PGPR can be employed in ecological and sustainable production of nursery material and point to the feasibility of synthetic auxin (IBA) replacement by PGPR-based ecological treatment. Acknowledgment The author would like to thank Prof. Dr. Ahmet Esitken for help in obtaining bacterial sources. References Ait-Barka, E., Nowak, J., Clément, C., 2006. Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans Strain PsJN. Appl. Environ. Microbiol. 72, 7246–7252. Alam, M.S., Cui, Z., Yamagishi, T., Ishi, R., 2003. Rice cultivar variation in the growth response to inoculation of free-living rhizobacteria. Plant Prod. Sci. 6, 50–51. Aslantas, R., Cakmakci, R., Sahin, F., 2007. Effect of plant growth promoting rhizobacteria on young apple tree growth and fruit yield under orchard conditions. Sci. Hortic. 111, 371–377. Baydar, N.G., Ece, M., 2005. Comparison of different scion/rootstock combinations in the production of grafted grapevines in Isparta condition. J. Sul. Dem. Univ. Nat. Sci. Enst. 9, 49–53. Bouquet, A., Hevin, M., 1978. Green-grafting between Muscadine grape (Vitis rotundifolia Michx.) and bunch grapes (Euvitis spp.) as a tool for physiological an pathological investigations. Vitis 17, 134–138. Compant, S., Reiter, B., Sessitsch, A., Nowak, J., Clement, E., Ait Barka, E., 2005. Endophytic colonization of Vitis vinifera L. by plant growth promoting bacterium Burkholderia sp. strain PsJN. Appl. Environ. Microbiol. 71, 1685–1693. Compant, S., Kaplan, H., Sessitsch, A., Nowak, J., Ait Barka, E., Clement, C., 2008. Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues. FEMS Microbiol. Ecol. 63, 84–93. Compant, S., Mitter, B., Colli-Mull, J.G., Gangl, H., Sessitsch, A., 2011. Endophytes of grapevine flowers, berries, and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microb. Ecol. 62, 188–197. Esitken, A., Karlidag, H., Ercisli, S., Turan, M., Sahin, F., 2003. The effects of spraying a growth promoting bacterium on the yield, growth and nutrient element composition of leaves of apricot (Prunus armeniaca L. cv. Hacihaliloglu). Aust. J. Agr. Res. 54, 377–380. Esitken, A., Pirlak, P., Turan, M., Sahin, F., 2006. Effects of floral and foliar application of plant growth promoting rhizobacteria (PGPR) on yield, growth of nutrition of sweet cherry. Sci. Hortic. 110, 324–327. Esitken, A., 2011. Use of plant growth promoting rhizobacteria in horticultural crops. In: Maheshwari, D.K. (Ed.), Bacteria in Agrobiology: Crop Ecosystems. Springer Pub, pp. 189–235. Frankenberger, W.T., Arshad, M., 1995. Phytohormones in Soils: Microbial Production and Function. Marcel Dekker, New York, NY. Gambetta, G.A., Rost, T.L., Matthews, M.A., 2009. Passive parhogen movement via open xylem conduits in grapevive graft unions. Am. J. Enol. Vitic. 60, 241–245.
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Gray, E.J., Smith, D.L., 2005. Intracellular and extracellular PGPR: commonalities and distinctions in the plant-bacterium signaling processes. Soil Biol. Biochem. 37, 3945–4412. Grichko, V.P., Glick, B.R., 2001. Amelioration of flooding stree by ACC deaminasecontaining plant growth-promoting bacteria. Plant Physiol. Biochem. 39, 11–17. Hartman, H.T., Kester, D.C., Davis, F.T., 1990. Plant Propagation Principles and Practices. Rajets/Prentice Hall, Inc., Englewood Cliffs, NJ, pp. 647. Karakurt, H., Aslantas, R., 2010. Effects of some plant growth promoting rhizobacteria (PGPR) strains on plant growth and leaf nutrient content of apple. J. Fruit Ornam. Plant Res. 18, 101–110. Kaserer, H., Leonhardt, W., Grahsl, A., Gangl, H., 2003. Report on efficient scheme to develop certified clonal propagating material in Austria. Acta Hortic. 603, 127–129. Kose, C., Guleryuz, M., Sahin, F., Demirtas, I., 2005. Effects of some plant growth promoting rhizobacteria (PGPR) on graft union of grapevine. J. Sustain. Agr. 26, 139–147. Koshita, Y., Takahara, T., Ogata, T., Goto, A., 1999. Involvement of endogenous plant hormones (IAA, ABA, GAs) in leaves and flower bud formation of satsuma mandarin (Citrus unshiu Marc.). Sci. Hortic. 79, 185–194. Lambrecht, M., Okon, Y., Broek, A.V., Vanderleyden, J., 2000. Indole-3-acetic acid a reciprocal signalling molecule in bacteria-plant interactions. Trends Microbiol. 298, 8–9. Lodewyckx, C., Vangronsveld, J., Porteous, F., Moore, E.R.B., Taghavi, S., Mezgeay, M., van der Lelie, D., 2002. Endophytic bacteria and their potential applications. Crit. Rev. Plant Sci. 21, 583–606. Maheshwari, D.K., 2011. Bacteria in Agrobiology: Crop Ecosystems. Springer, Heidelberg/Dordrecht/London/New York, pp. 434. Martelli, G.P., Savino, V., Walter, B., 1993. Indexing on Vitis indicators. In: Martelli, G.P. (Ed.), Graft Transmissible Diseases of Grapevines. Food and Agriculture Organization of the United Nations, Rome, pp. 137–155. Pathirana, R., McKenzie, M.J., 2005. A modified green-grafting technique for largescale virus indexing of grapevine (Vitis vinifera L.). Sci. Hortic. 107, 97–102. Piccolo, S., Ferrano, V., Alfonzo, A., Settanni, L., Ercolini, D., Burruano, S., Moshetti, G., 2010. Presence of endophytic bacteria in Vitis vinifera leaves as detected by fluorescence in situ hybridization. Ann. Microbiol. 60, 161–167. Rosenblueth, M., Martinez-Romero, E., 2006. Bacterial endophytes and their interactions with hosts. Mol. Plant-Microbe Interact. 19, 827–837. Sabir, A., Kara, Z., 2004. Performance and success of green grafting technique in some American rootstocks. Alatarim 3, 28–32. Sabir, A., Agaoglu, Y.S., 2009. The effects of different IBA and NAA applications on grafting success of some cultivar/rootstock combinations in potted grape sapling production. Alatarim 8, 22–27. Sabir, A., 2011. Comparison of green grafting techniques for success and vegetative development of grafted grape cultivars (Vitis spp.). Int. J. Agric. Biol. 13, 628–630. Sabir, A., Yazici, M.A., Kara, Z., Sahin, F., 2012. Growth and mineral acquisition response of grapevine rootstocks (Vitis spp.) to inoculation with different strains of plant growth-promoting rhizobacteria (PGPR). J. Sci. Food Agric. 92, 2148–2153. Ulger, S., Sonmez, S., Karkacier, M., Ertoy, N., Akdesir, O., Aksu, M., 2004. Determination of endogenous hormones, sugars and mineral nutrition levels during the induction, initiation and differentiation stage and their effects on flower formation in olive. Plant Growth Regul. 42, 89–95. Walker, M.A., Meredith, C.P., 1990. The genetics of resistance to grapevine fanleaf virus (GFV) in Vitis vinifera. Vitis 1990, 228–238. Walter, B., Bass, P., Legin, R., Martin, C., Vernoy, R., Collas, A., Vesselle, G., 1990. The use of a green–grafting technique for the detection of virus–like diseases of the grapevine. J. Phytopathol. 128, 137–145. Yang, J., Kloepper, J.W., Ryu, C.M., 2009. Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci. 14, 1–4. Yin, H., Yan, B., Sun, J., Jia, P., Zhang, Z., Yan, X., Chai, J., Ren, Z., Zheng, G., Liu, H., 2012. Graft-union development: a delicate process that involves cell–cell communication between scion and stock for local auxin accumulation. J. Exp. Bot. 63, 4219–4232.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Improvement of grafting efficiency in hard grafting grape Berlandieri hybrid rootstocks by plant growth-promoting rhizobacteria (PGPR) Ali Sabir ∗ University of Selcuk, Faculty of Agriculture, Department of Horticulture, 42075 Konya, Turkey
a r t i c l e
i n f o
Article history: Received 30 May 2013 Received in revised form 22 August 2013 Accepted 23 August 2013 Keywords: Green grafting Callus formation Graft survival PGPR
a b s t r a c t Certain grapevine rootstocks in Vitis berlandieri hybrids (such as 41 B, one of the most important rootstocks used in worldwide viticulture) have great potential to use owing to its great resistance to calcareous soils and phylloxera. But their use is limited due to the difficulties in propagation and grafting. PGPR is reported to stimulate division and enlargement of plant cells that might result in better fusion at graft union point. This investigation was thus conducted to reveal the effects of plant growth promoting rhizobacteria (Pseudomonas putida strain BA-8, Bacillus simplex strain T7) on callus formation, graft success, vegetative development and subsequent bud fruitfullness of Alphonse Lavallée grape cultivar. The green softwood shoots of Alphonse Lavallée (AL) grape were grafted on the rootstocks 41 B or 1103 P by modified cleft green-grafting technique in controlled glasshouse. Before grafting, the single scion nodes were divided into four treatment groups. Three of them were separately dipped into bacterial suspension (109 CFU ml−1 ) of either of two strains or their combination for an hour. One of the treatment groups was dipped into the tap water (as control). Each treatment has contained thirty grafts, divided into three replicates. In both graft combinations, combined application of BA-8 and T7 strains resulted in 100% callusing compared with the uninoculated control where the percentages were 93.3 and 90.0% for AL/41 B and AL/1103 P combinations, respectively. Individual or combined applications of the strains also had remarkably positive influences on full callusing rates. The highest shoot length values were obtained from combined application (113.5 cm) and BA-8 strains (100.9 cm) in AL/41 B and AL/1103 P graft combinations, respectively. Inoculation of the graft scion parts with BA-8 strain resulted in the highest shoot lignification lengths in both AL/41 B and AL/1103 P combinations with the values of 75.4 and 75.6 cm, respectively while uninoculated grafts of the representing combinations were as low as 59.0 and 55.3 cm. In AL/41B, the highest (100%) graft survival rate in nursery was recorded in plants treated with bacterial combination. Combined application as well as BA-8 performed better in AL/1103P. The findings imply that PGPR can be employed in ecological and sustainable production of nursery material and point to the feasibility of commercially synthetic auxin replacement by PGPR-based ecological treatment. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In modern cropping systems, grafting is commonly used to impart pathogen resistance, manipulate grapevine physiology, confer disease or pest resistance, and tolerate certain soil types (Gambetta et al., 2009). Grape rootstocks have been bred to provide resistance to improper soil condition, diseases such as phylloxera (Daktulosphaira vitifoliae Fitch) or other pathological problems. The practice of grafting onto disease-resistant stocks now extends to a variety of horticultural plants. However, mistakes in any stages of grafting can result in serious problems difficult to compensate. Many factors may affect the success of grafting such as time of
∗ Tel.: +90 332 223 29 09; fax: +90 332 241 01 08. E-mail address: [email protected] 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.08.035
grafting, cold treatment of the cuttings and hormonal application. Anatomically, the cambium of a stock and scion must be in close contact to form a union. During the grafting process, undifferentiated callus cells are formed, which will differentiate into specialized cells that form a new xylem (water and nutrient pathway) and phloem (sugar pathway) within the graft union. Several different grafting techniques can be performed to generate complete grapevines. Among them, green grafting offers growers to propagate the desired cultivars during the growing period after the time for grafting with dormant scions has ended. In this technique, succulent green current-season growth is used as scions that are grafted on the new growth of cultivar rootstock. Furthermore, green grafting can be performed early as shoots are large and firm enough to handle. The method has been successfully adopted in many countries, where grape cultivation is common (Kaserer et al., 2003). Green grafting technique has many
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other advantages such as easiness for application, relatively more efficient graft-scion affinity to overcome the graft incompatibility sometimes experienced between distantly related Vitis species (Bouquet and Hevin, 1978; Sabir and Kara, 2004). This technique is currently being used for rapid and routine indexing of grapevine virus diseases (leafroll, fleck, corky bark, vein mosaic, vein necrosis) and more recently as a means of evaluating virus presence in certification programs (Walker and Meredith, 1990; Walter et al., 1990; Pathirana and McKenzie, 2005). Currently, application of concrete grafting types has gained importance for obtaining successful result, although little data exist regarding the advantages and disadvantages of different green-grafting methods (Sabir, 2011). Some grapevine rootstocks such as Vitis berlandieri have great potential to be used as grape rootstock owing to its resistance to calcareous soils and phylloxera, but their use is limited due to the difficulties in propagation and grafting. This is a common problem in rooting or grafting of 41 B rootstock although it is one of the most important rootstocks used around the world. For a successful propagation, increasing callus formation, grafting union, and grafting success are very important, and PGPR can have effects on success rate (Maheshwari, 2011). The effectiveness of grafting zone inoculation with Pseudomonas BA-8 (cytokinin producer) and Bacillus BA16 and OSU-142 (IAA producers) to improve successing rate, callusing rate, callusing degree, and full callusing rate on four different scion-rootstock combinations were studied by Kose et al. (2005). All the strains significantly increased success rate of grafting in varying degrees among the grafting combinations compared to the noninoculated control. Some of such PGPR classified as endophytes are able to transcend the endodermis barrier, crossing from the root cortex to the vascular system, and subsequently thrive as endophytes in stem, leaves, tubers, and other organs (Compant et al., 2005). The extent of endophytic colonization of host plant organs and tissues reflects the ability of bacteria to selectively adapt to these specific ecological niches (Gray and Smith, 2005; Esitken et al., 2006). As a result, intimate associations between bacteria and host plants can be formed without harming the plant (Lodewyckx et al., 2002). PGPR colonize the plant rhizosphere and positively affect their growth and responses to stress (Compant et al., 2008; Yang et al., 2009). In general, PGPR treatments were given to roots, but recently, sprayed to aerial part of plants (Esitken, 2011). To our knowledge, surprisingly, very little is known about the effects of PGPR on graft success in grapevine as well as other perennials, except for a few studies (Kose et al., 2005). Therefore, this investigation was conducted to evaluate the effects of plant growth promoting rhizobacteria (Bacillus simplex T7 and Pseudomonas putida strain BA-8) on callus formation, graft success and early development of scion shoots of Alphonse Lavallée grape cultivar on two different rootstocks 41 B (very difficult to root with a low graft success) and 1103 P (moderate rooting and grafting success) using a relatively new and high-success grafting type.
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on the rootstocks by modified cleft green-grafting technique. The green softwood shoots chosen for grafting had an equal diameter to the rootstocks (5–8 mm), and were at 3–6 nodes below the active bud (Walter et al., 1990). Modified cleft grafting was applied as described previously by Sabir (2011). A slit was made down the center of the scion by a razor blade and a long tapered wedge was made on the opposing piece (rootstock). For all grafts, the shoots of both rootstock and the scion had the same diameter at the point of graft contact so that the cambium layers of the union matched (Martelli et al., 1993). The assembled grafts were wrapped with commercial plastic bandage tape. Each rootstock plant had one graft scion and the remaining shoots on grafted plants were discarded. 2.2. Bacterial inoculum and plant bacterization The grafted plants were randomly divided into 4 treatment groups. Three groups of the prepared scion parts were separately immersed into individual or combined bacterial solutions of cytokinin synthesizing Pseudomonas putida strain BA-8 and auxin synthesizing Bacillus simplex strain T7 (109 CFU ml−1 ) in 2 l containers and incubated at room temperature for an hour. One of the treatment groups was dipped into the tap water and used as a control. After treatments, grafting was performed immediately by using modified cleft method. For each bacterial treated and control groups, two different combinations of rootstock-scion including Alphonse Lavallée/41 B and Alphonse Lavallée/1103 P were used. 2.3. Growth condition The graft union was healed under condition of moderate light, fairly high humidity (between 60 and 80%) and good air circulation. The grafted plants were kept under tulle curtain having 50% transmission, with mean daily temperature in the glasshouse ranged between 21 and 30 ◦ C (according to the temperature and humidity data logger) during the experiment over 3 weeks. Then, the curtain was removed to allow the scion shoot to harden properly. Night and day temperatures during the vegetative development period were 20 ± 3 and 28 ± 4 ◦ C respectively. To achieve a concrete irrigation, two soil moisture sensors (Irrometer company, Riverside, CA) were installed 30 cm depths to continuously monitor changes in soil–water content (http://www.irrometer.com). The irrometer readings were taken every day to maintain the soil moisture at the field capacity level according to the manufacturer’s guideline. To ensure the uniformity of irrigation, the water was transported directly into the pots by micro irrigation systems consisted of individual spaghetti tubes. Lateral shoots emerging from the active summer buds of plants were removed daily, allowing only the main shoots to elongate in order to ensure valid comparison of development. The shoots were tied with thread to wires established 2.5 m above the pots to let the plants grow on a fence in an upright position, thus ensuring equal benefit from the light. 2.4. Plant analyses
2. Materials and methods 2.1. Plant material and graft operation The study was established in an experimental glasshouse of Selcuk University, Agriculture Faculty, Horticulture Department, Konya, Turkey. One year old own rooted 41 B (Vinifera x Berlandieri) and 1103 P (Berlandieri x Rupestris) vines in equal vegetative growth were individually cultivated in 8 l plastic pots containing uniform mixture of peat (1.034% N, 0.94% P2 O5 , 0.64% K2 O, pH 5.88, Klassman® ) and inert perlite (0–3 mm in diameter) in equal volume. After a two-month vegetative development stage in early spring of 2012, grapevine cultivars Alphonse Lavallée was grafted
For the analyses of callusing response of the grafts to bacterization, graft unions were visually evaluated 4 weeks after grafting operation for callusing rate, full callusing rate and callusing degree as described previously by Hartman et al. (1990), Baydar and Ece (2005) and Sabir (2011). Callusing rate was determined as percentage by using callus formation around the graft cut surfaces. Full callusing rate was the percentage of grafts of which graft cut surfaces callused at 100%. Callus degree was determined by using 1–4 scale; 1: 1–25% callusing, 2: 26–50% callusing, 3: 51–75% callusing and 4: 76–100% callusing at graft union point. In order to investigate the growth promoting effect of bacterization, measurements on shoot length (scion shoot was measured with a sensitivity of 1 mm),
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A. Sabir / Scientia Horticulturae 164 (2013) 24–29
Fig. 1. Early development course of AL/41 B with respect to bacteria applications. Error bar stands for the standard deviation of the means.
Fig. 2. Early development course of AL/1103 P with respect to bacteria applications. Error bar stands for the standard deviation of the means.
shoot lignification length (the length of the scion shoot, where complete lignification occurred) and shoot diameter (measured by digital compass at a point 1 cm above the second node). Early shoot development was also investigated by measurements performed with 2–4 days intervals. Besides, fruitfulness response of bacterized grafts was investigated at the beginning of the growth season of the following year (2013). For this, each plant in glasshouse was pruned back at the end of the winter season after experimental growth season leaving two winter buds per plant (considering the genetic bud fruitfulness potential of Alphonse Lavallée cultivar). After bud sprout and subsequent two-month-shoot development, clusters born on the one-year-old grafts were calculated.
apparent in the grafts. The response of different graft combinations to bacterial inoculums differed with respect to early development of scion shoots. Individual or combined applications of the bacteria strains significantly (P < 0.05) increased the scion shoot length, shoot lignification length and shoot diameter of all the graft combinations on both the rootstocks (Table 2). The highest shoot length values were obtained from combined application (113.5 cm) and BA-8 strains (100.9 cm) in AL/41 B and AL/1103 P graft combinations, respectively. Inoculation of the graft scion parts with BA-8 strain resulted in the highest shoot lignification lengths among the AL/41 B and AL/1103 P combinations with the values of 75.4 and 75.6 cm, while uninoculated grafts of the representing combinations were as low as 59.0 and 55.3 cm, respectively. Shoot diameter response of the grafts were also in accordance with the results obtained from shoot lignification length. The grafts inoculated with the stains of BA-8 had the highest shoot diameter values in both graft combinations. As can be seen in Table 3, all bacterial inoculations increased the leaf (node) number across the grafts. In particular, the most effective application was combined strains. To illustrate, the highest values of leaf number per plant were 25.3 and 26.7 for AL/41 B and AL/1103 P combinations, while uninoculated controls had 20.0 and 21.5 leaves per plant, respectively. Individual treatments of bacteria strains had little or no effect on leaf area of the grapevines. However, combined applications significantly (P < 0.05) increased the leaf area in AL/41 B and AL/1103 P grafts with their values 95.4 and 91.7 cm2 while the controls of the same grafts had 86.4 and 82.7 cm2 , respectively. Pruning residue was also a good indicator of the response of grapevine grafts to inoculation with bacteria strains. Inoculation induced an enhancement of shoot pruning residue weight as well as the number of nodes. As presented in Fig. 3, in AL/41B, the highest (100%) graft survival rate was recorded in plants treated with bacterial combination. Also, combined application as well as BA-8 was better in AL/1103P. Bacteria strains clearly enhanced the graft success, although their effects were cultivar-dependent.
2.5. Statistical analysis The study design was a complete randomized block with three replicates. Each replicate consisted of 10 grafted plants. Data for each rootstock were separately evaluated by analysis of variance (ANOVA) and treatment means were separated by Least Significant Differences (LSD) test at P < 0.05. Data were analyzed using SPSS program version 13.0 (SPSS Inc., Chicago, IL). 3. Results 3.1. Callusing response of the grafts to bacterization Inoculation of PGPR strains alone or in combination significantly (P < 0.05) increased callusing rate, full callusing rate and callusing degree accross the graft combinations, except for callusing rate in Alphonse Lavallée (AL)/41 B (Table 1). In both graft combinations, combined application of Pseudomonas BA-8 and Bacillus simplex T7 strains resulted in 100% callusing compared with the uninoculated control where the percentages were 93.3 and 90.0% for AL/41 B and AL/1103 P, respectively. Similarly, bacteria strains significantly (P < 0.05) improved full callusing rates in grafts, with the highest rates obtained from combined application across the grafts. As for the callusing degree around the graft union point, individual or combined applications of the strains had also remarkably positive influences with the highest degrees being promoted by combined application in AL/41 B (3.84) and BA-8 strains in AL/1103 P (3.95). 3.2. Growth promoting effect of bacterization In the scope of comparing early shoot developments among the experimental groups, shoot length measurement performed with 2–4 days intervals in early development stage revealed that bacteria applications had significantly (P < 0.05) positive effect on AL/41 B grafts (Fig. 1) and slight effect on AL/1103 P combination (Fig. 2). Positive effects of combined application or BA-8 alone were more
Fig. 3. Survival rate responses of 41 B and 1103 P grafts to bacterization. Each column represents the mean of triplicate determinations with ten grafts for replicate. Error bar stands for the standard deviation of that mean.
A. Sabir / Scientia Horticulturae 164 (2013) 24–29
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Table 1 Effects of bacterial strains on callus formation of different graft combinations. Scion/rootstock
Inoculant
Callusing rate (%)
Full callusing rate (%)
Callusing degree (1–4)
AL/41B
Control T7 BA-8 T7+BA-8
93.3 ± 5.77 100 ± 0.00 96.7 ± 5.77 100 ± 0.00
56.7 ± 5.77 c 73.3 ± 5.77 b 70.0 ± 0.00 bc 86.7 ± 5.77 a
3.51 ± 0.08 c 3.74 ± 0.06 ab 3.67 ± 0.11 bc 3.84 ± 0.04 a
ns
10.8
0.14
90.0 ± 0.00 b 96.7 ± 5.77 a 100 ± 0.00 a 100 ± 0.00 a
60.0 ± 0.00 b 76.7 ± 5.77 a 83.3 ± 5.77 a 90.0 ± 0.00 a
3.38 ± 0.20 b 3.90 ± 0.09 a 3.95 ± 0.08 a 3.85 ± 0.14 a
5.52
7.65
0.25
LSD (%5) AL/1103P
Control T7 BA-8 T7+BA-8
LSD (%5)
Levels not connected by same letter are significantly different; AL, Alphonse Lavallée; and ns, not significant.
Table 2 Effects of bacterial strains on shoot developments of different graft combinations. Scion/rootstock
Inoculant
Length (cm)
Lignification length (cm)
Diameter (mm)
AL/41B
Control T7 BA-8 T7+BA-8
73.1 ± 2.47 d 82.9 ± 3.35 c 95.1 ± 2.71 b 113.5 ± 7.07 a
59.0 ± 5.51 b 57.9 ± 0.90 b 75.4 ± 2.25 a 75.2 ± 3.61 a
4.03 ± 0.10 c 4.17 ± 0.04 b 4.48 ± 0.06 a 4.37 ± 0.05 a
8.1
6.60
0.12
84.7 ± 4.73 b 96.3 ± 3.81 a 100.9 ± 2.83 a 100.3 ± 7.76 a
55.3 ± 3.77 b 71.9 ± 1.90 a 75.6 ± 4.00 a 74.8 ± 3.38 a
4.39 ± 0.03 c 4.34 ± 0.05 c 4.65 ± 0.03 b 4.72 ± 0.02 a
9.63
6.30
0.07
LSD (%5) AL/1103P
Control T7 BA-8 T7+BA-8
LSD (%5)
Levels not connected by same letter are significantly different; AL, Alphonse Lavallée; and ns, not significant.
Table 3 Effects of bacterial strains on leaf development and pruning residue of different graft combinations. Scion/rootstock
Inoculant
Leaf (node) number
Leaf area (cm2 )
Pruning residue (g)
AL/41B
Control T7 BA-8 T7+BA-8
20.0 ± 0.23 c 20.3 ± 0.25 c 23.4 ± 0.81 b 25.3 ± 0.78 a
86.4 ± 1.65 b 93.4 ± 1.76 a 85.9 ± 2.49 b 95.4 ± 2.64 a
7.53 ± 0.15 c 7.43 ± 0.12 c 9.89 ± 0.00 b 11.88 ± 0.43 a
1.10
4.09
0.45
21.5 ± 0.46 c 24.2 ± 0.20 b 25.6 ± 0.95 a 26.7 ± 0.46 a
82.7 ± 3.30 b 82.5 ± 1.68 b 84.3 ± 1.62 b 91.7 ± 2.25 a
9.87 ± 0.45 c 11.65 ± 0.55 b 13.47 ± 0.40 a 13.24 ± 0.21 a
1.10
4.05
0.78
LSD (%5) AL/1103P
Control T7 BA-8 T7+BA-8
LSD (%5)
Levels not connected by same letter are significantly different; (mean ± SD); AL, Alphonse Lavallée; and ns, not significant.
3.3. Fruitfulness response of bacterized grafts According to the findings obtained in the beginning of the following vegetation period, all strains significantly (P < 0.05) increased the number of cluster per plant (Fig. 4) across the grafts on both 41 B and 1103 P, compared with the uninoculated control, the maximum increase being promoted by BA-8 in 41 B and combined treatment in 1103 P. In AL/41 B grafts, BA-8 induced more than two fold increment in cluster number per young vines while a very similar inducement was obvious in combined treatment of AL/1103 P.
agricultural techniques and bio-fertilization are vital to alleviating environmental pollution. Over the past two decades, research on bacteria species has been steadily expanding as the strains of various bacteria ecologically enhance plant growth, disease
4. Discussion Intensive agriculture still relies on the use of chemical fertilizers to provide faster vegetative growth and high fruit yield although excessive use of chemical fertilizers causes problems not only in terms of financial cost but also in terms of the cost to the environment. Therefore, the development and application of sustainable
Fig. 4. Cluster development responses of 41 B and 1103 P grafts to bacterization. Each column represents the mean of triplicate determinations with ten grafts for replicate. Error bar stands for the standard deviation of that mean.
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resistance, and improve nutrient fixation or solution (Ait-Barka et al., 2006; Karakurt and Aslantas, 2010). Such findings have stimulated a growing interest for using PGPR isolates in viticulture for various purposes (Esitken, 2011). However might it very be surprising, literature data to investigate the effects of PGPR strains on graft success increment in Vitis species is insufficient although it has been very well-known that certain bacteria species produce auxin and/or auxin like plant growth promoting substances (Kose et al., 2005). In the present study, the bacteria strains Pseudomonas BA-8 (cytokinin producer) and Bacillus simplex T7 (auxin producer) stimulated the formation of callus tissue around the graft point. Combined application of the bacteria strain had more pronounced positive effect on callus formation parameters in AL/41 B while BA-8 was withstanding with its individual positive effect in AL/1103 P. This finding is in accordance with the findings of Aslantas et al. (2007) who observed higher IAA level produced by BA-8 among three different strains (Pseudomonas BA8, Bacillus BA16 and Bacillus OSU142) by HPLC detections. Also, previous studies verified that strains in the genus studied in the present study Pseudomonas and Bacillus were able to produce plant growth regulators particularly indole-3-acetic acid (IAA) (Lambrecht et al., 2000). According to the tissue analyze studies, an increasing number of IAA-producing plant growth-promoting rhizobacteria (PGPR) are detected inside the plant tissue in relation with the amount of bacteria (Rosenblueth & Martinez-Romero, 2006). In addition to this, Grichko and Glick (2001) set forth that Pseudomonas strains may have ACC deaminase enzyme which can cleave the plant ethylene precursor ACC and thereby lower the level of ethylene in plants. Adhesion between the rootstock and the scion is aided by a socalled tissue “callus”. Wound-repair xylem is generally the first differentiated tissue to bridge the graft union. Aside from whatever role IAA plays in promoting cell division (callus formation) during graft union formation (Yin et al., 2012), IAA and cytokinin plays an important role in xylem formation across the callus bridge between stock and scion. Further, cambial continuity after callusing between graft partners has also an essential role in graft survival rate (Sabir, 2011). From a practical perspective, however, exogenous application of auxin in grafting has not caught on, because the response of Vitis materials is small, if not, and variable (Sabir and Agaoglu, 2009). Therefore the bacterial source of IAA and cytokinin application to promote formation of new vascular tissue promises potential and sustainable advances. Previously Esitken (2011) stated that PGPR, with their various hormonal potentials, can be used for various purposes such as rooting of cutting, grafting union, fruit setting and thinning, lateral root formation, increasing tolerance against abiotic stress as well as growth, development, and biological control with root inoculation and/or spraying. The growth inducing effects of the bacteria strains revealed in the present study verifies the previous findings obtained by Esitken et al. (2003) in apricots and Aslantas et al. (2007) in apples where inducing effects of especially BA-8 strains on shoot growth were documented. Among the physiological mechanisms pertaining to vegetative growth inducement, according to the literature, the most important one might be related to bacterial production of the phytohormone IAA (Frankenberger and Arshad, 1995). Studies also indicated that the extent of positive effects of various bacterial strains on plant growth varied with the species or variety of host plant (Alam et al., 2003; Kose et al., 2005; Sabir et al., 2012), as seen in this study with different response of grafts. In this study, early shoot development of bacteria inoculated plants were also better than those of control ones. This outcome supports the results of a previous study that reveals the movement of vascular bacteria between scion and rootstocks (Gambetta et al., 2009). The researchers demonstrated that the bacteria moves through open xylem conduits of grapevine graft union and may carry out systemic
infection in vine tissues. Using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM), Piccolo et al. (2010) and Compant et al. (2011) also detected the distribution of various bacterial colonies in seeds, flowers, leaf veins, cells, hairs, intercellular spaces, and cut edges of leaf disks of five different grape cultivars. They further identified the bacteria strains such as Bacillus spp., Burkholderia spp., Pseudomonas spp., in many samples by 16S rRNA gene sequencing or microscopic investigations. Ait-Barka et al. (2006) demonstrated that PGPR Burkholderia phytofirmans strain PsJN increased grapevine growth and physiological activity even at low temperatures. The mentioned literature, including the present findings, suggests the potential role of bacteria as plant growth promoters and sources of plant growth manipulating substances. Apart from the success in graft and callus formation, survival rate in nursery and early growth in vineyard is essential for a profitable use of agricultural area (Sabir et al., 2012). In the current study, bacteria inoculation alone or in combination markedly helped the young plants to survive. Previously, using different hardwood grafting type and varieties, Kose et al. (2005) demonstrated that there were statistically important increases in the survival of grafts in relation with PGPR applications. Previous report set fort that the PGPR stick to plant cell within an hour after inoculation and migrate into the entire plant parts including leaves and inflorescences (Compant et al., 2005, 2008) and then an interaction between plant tissue and the bacteria is established. The sustained growth responses in both vegetative and generative aspects investigated in this study seem to be in accordance with the reports mentioned above. Bacterized graft shoots not only grew faster than nonbacterzed controls but also were sturdier, with a better developed thick shoots. This, combined with an enlarged leaf growth may have contributed to the stimulation of generative development, namely floral changes which occur during early shoot development stage in buds. Because, the plants having much more morphological development under the bacteria effects were also having significantly greater cluster numbers born in the following year of this study. Previous studies indicated that the high concentrations of cytokinin and IAA levels in leaves and nodes of perennial plants during the induction and initiation of floral periods, may physiologically promote flower formation (Koshita et al., 1999; Ulger et al., 2004). These literature and the present results verify that the bacterial inoculation resulted in better floral development with their growth inducing effects emerging from consolidated hormone levels in newly grafted plants.
5. Conclusion Overall investigations in the present study indicated that individual and/or combined applications of the strains of BA-8 and T7 have significantly positive influences on graft callusing, scion shoot growth, cane hardening, and nursery survival rate as well as fruitfulness of the grapes in following year. Combined application of bacteria strains displayed more pronounced effects on most of the investigated criteria. Callus promoting potential of the strains clarified their ability to synthesize plant growth regulators. The findings of current study is anticipated to shed light into the application and sustainable use of bacterial bioinoculants in enhancing graft success, survival rate, nursery and young vineyard development as well as fruitfulness and cold hardening of grapevines. Considering the large lose in acclimatization stage of intact- and nursery-grown grapevines, especially when 41 B rootstock is used, the employment of PGPR promises positive effects on enhancement of graft success. Therefore, further studies may be useful to sucrutinize the cultivar-specific activities of PGPR with
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physiological and anatomic mechanisms underlying the investigated growth. Most of the viticulture area on the world today face with serious problems emerging from biotic or abiotic stress factors such as phylloxera, nematodes, water shortage, winter cold, excessive limestone, etc. Therefore, practical application studies on the sustainable use of such PGPRs under stress condition will be our next step toward a better benefiting the role of microorganisms. The results finally imply that such PGPR can be employed in ecological and sustainable production of nursery material and point to the feasibility of synthetic auxin (IBA) replacement by PGPR-based ecological treatment. Acknowledgment The author would like to thank Prof. Dr. Ahmet Esitken for help in obtaining bacterial sources. References Ait-Barka, E., Nowak, J., Clément, C., 2006. Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans Strain PsJN. Appl. Environ. Microbiol. 72, 7246–7252. Alam, M.S., Cui, Z., Yamagishi, T., Ishi, R., 2003. Rice cultivar variation in the growth response to inoculation of free-living rhizobacteria. Plant Prod. Sci. 6, 50–51. Aslantas, R., Cakmakci, R., Sahin, F., 2007. Effect of plant growth promoting rhizobacteria on young apple tree growth and fruit yield under orchard conditions. Sci. Hortic. 111, 371–377. Baydar, N.G., Ece, M., 2005. Comparison of different scion/rootstock combinations in the production of grafted grapevines in Isparta condition. J. Sul. Dem. Univ. Nat. Sci. Enst. 9, 49–53. Bouquet, A., Hevin, M., 1978. Green-grafting between Muscadine grape (Vitis rotundifolia Michx.) and bunch grapes (Euvitis spp.) as a tool for physiological an pathological investigations. Vitis 17, 134–138. Compant, S., Reiter, B., Sessitsch, A., Nowak, J., Clement, E., Ait Barka, E., 2005. Endophytic colonization of Vitis vinifera L. by plant growth promoting bacterium Burkholderia sp. strain PsJN. Appl. Environ. Microbiol. 71, 1685–1693. Compant, S., Kaplan, H., Sessitsch, A., Nowak, J., Ait Barka, E., Clement, C., 2008. Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues. FEMS Microbiol. Ecol. 63, 84–93. Compant, S., Mitter, B., Colli-Mull, J.G., Gangl, H., Sessitsch, A., 2011. Endophytes of grapevine flowers, berries, and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microb. Ecol. 62, 188–197. Esitken, A., Karlidag, H., Ercisli, S., Turan, M., Sahin, F., 2003. The effects of spraying a growth promoting bacterium on the yield, growth and nutrient element composition of leaves of apricot (Prunus armeniaca L. cv. Hacihaliloglu). Aust. J. Agr. Res. 54, 377–380. Esitken, A., Pirlak, P., Turan, M., Sahin, F., 2006. Effects of floral and foliar application of plant growth promoting rhizobacteria (PGPR) on yield, growth of nutrition of sweet cherry. Sci. Hortic. 110, 324–327. Esitken, A., 2011. Use of plant growth promoting rhizobacteria in horticultural crops. In: Maheshwari, D.K. (Ed.), Bacteria in Agrobiology: Crop Ecosystems. Springer Pub, pp. 189–235. Frankenberger, W.T., Arshad, M., 1995. Phytohormones in Soils: Microbial Production and Function. Marcel Dekker, New York, NY. Gambetta, G.A., Rost, T.L., Matthews, M.A., 2009. Passive parhogen movement via open xylem conduits in grapevive graft unions. Am. J. Enol. Vitic. 60, 241–245.
29
Gray, E.J., Smith, D.L., 2005. Intracellular and extracellular PGPR: commonalities and distinctions in the plant-bacterium signaling processes. Soil Biol. Biochem. 37, 3945–4412. Grichko, V.P., Glick, B.R., 2001. Amelioration of flooding stree by ACC deaminasecontaining plant growth-promoting bacteria. Plant Physiol. Biochem. 39, 11–17. Hartman, H.T., Kester, D.C., Davis, F.T., 1990. Plant Propagation Principles and Practices. Rajets/Prentice Hall, Inc., Englewood Cliffs, NJ, pp. 647. Karakurt, H., Aslantas, R., 2010. Effects of some plant growth promoting rhizobacteria (PGPR) strains on plant growth and leaf nutrient content of apple. J. Fruit Ornam. Plant Res. 18, 101–110. Kaserer, H., Leonhardt, W., Grahsl, A., Gangl, H., 2003. Report on efficient scheme to develop certified clonal propagating material in Austria. Acta Hortic. 603, 127–129. Kose, C., Guleryuz, M., Sahin, F., Demirtas, I., 2005. Effects of some plant growth promoting rhizobacteria (PGPR) on graft union of grapevine. J. Sustain. Agr. 26, 139–147. Koshita, Y., Takahara, T., Ogata, T., Goto, A., 1999. Involvement of endogenous plant hormones (IAA, ABA, GAs) in leaves and flower bud formation of satsuma mandarin (Citrus unshiu Marc.). Sci. Hortic. 79, 185–194. Lambrecht, M., Okon, Y., Broek, A.V., Vanderleyden, J., 2000. Indole-3-acetic acid a reciprocal signalling molecule in bacteria-plant interactions. Trends Microbiol. 298, 8–9. Lodewyckx, C., Vangronsveld, J., Porteous, F., Moore, E.R.B., Taghavi, S., Mezgeay, M., van der Lelie, D., 2002. Endophytic bacteria and their potential applications. Crit. Rev. Plant Sci. 21, 583–606. Maheshwari, D.K., 2011. Bacteria in Agrobiology: Crop Ecosystems. Springer, Heidelberg/Dordrecht/London/New York, pp. 434. Martelli, G.P., Savino, V., Walter, B., 1993. Indexing on Vitis indicators. In: Martelli, G.P. (Ed.), Graft Transmissible Diseases of Grapevines. Food and Agriculture Organization of the United Nations, Rome, pp. 137–155. Pathirana, R., McKenzie, M.J., 2005. A modified green-grafting technique for largescale virus indexing of grapevine (Vitis vinifera L.). Sci. Hortic. 107, 97–102. Piccolo, S., Ferrano, V., Alfonzo, A., Settanni, L., Ercolini, D., Burruano, S., Moshetti, G., 2010. Presence of endophytic bacteria in Vitis vinifera leaves as detected by fluorescence in situ hybridization. Ann. Microbiol. 60, 161–167. Rosenblueth, M., Martinez-Romero, E., 2006. Bacterial endophytes and their interactions with hosts. Mol. Plant-Microbe Interact. 19, 827–837. Sabir, A., Kara, Z., 2004. Performance and success of green grafting technique in some American rootstocks. Alatarim 3, 28–32. Sabir, A., Agaoglu, Y.S., 2009. The effects of different IBA and NAA applications on grafting success of some cultivar/rootstock combinations in potted grape sapling production. Alatarim 8, 22–27. Sabir, A., 2011. Comparison of green grafting techniques for success and vegetative development of grafted grape cultivars (Vitis spp.). Int. J. Agric. Biol. 13, 628–630. Sabir, A., Yazici, M.A., Kara, Z., Sahin, F., 2012. Growth and mineral acquisition response of grapevine rootstocks (Vitis spp.) to inoculation with different strains of plant growth-promoting rhizobacteria (PGPR). J. Sci. Food Agric. 92, 2148–2153. Ulger, S., Sonmez, S., Karkacier, M., Ertoy, N., Akdesir, O., Aksu, M., 2004. Determination of endogenous hormones, sugars and mineral nutrition levels during the induction, initiation and differentiation stage and their effects on flower formation in olive. Plant Growth Regul. 42, 89–95. Walker, M.A., Meredith, C.P., 1990. The genetics of resistance to grapevine fanleaf virus (GFV) in Vitis vinifera. Vitis 1990, 228–238. Walter, B., Bass, P., Legin, R., Martin, C., Vernoy, R., Collas, A., Vesselle, G., 1990. The use of a green–grafting technique for the detection of virus–like diseases of the grapevine. J. Phytopathol. 128, 137–145. Yang, J., Kloepper, J.W., Ryu, C.M., 2009. Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci. 14, 1–4. Yin, H., Yan, B., Sun, J., Jia, P., Zhang, Z., Yan, X., Chai, J., Ren, Z., Zheng, G., Liu, H., 2012. Graft-union development: a delicate process that involves cell–cell communication between scion and stock for local auxin accumulation. J. Exp. Bot. 63, 4219–4232.