Generation and Characterization of Transgenic Plum Lines Expressing gafp-1 with the bul409 Promoter

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  • 1 Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson, SC 29634
  • | 2 USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430

The Gastrodia antifungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously generated, transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were created from seeds derived from open pollination of the cultivar Bluebyrd (BB-OP) with gafp-1 under the control of the polyubiquitin promoter bul409 and evaluated for Phytophthora root rot (PRR) and Root knot nematode (RKN) susceptibility. One of nine transgenic lines synthesizing GAFP-1 exhibited increased tolerance to PRR caused by P. cinnamomi. The same line (BB-OP-1) was also significantly more tolerant to RKN infection caused by Meloidogyne incognita. BB-OP-1 was more resistant to PRR and equally resistant to RKN compared with the cultivar Stanley-derived 4J line, which expresses gafp-1 under the control of the 35SCaMV promoter. GAFP-1 synthesis in BB-OP-1 was not elevated by pathogen infection, suggesting that the bul409 promoter is not inducible in the plum/GAFP-1 system. This study confirms the usefulness of the gafp-1 gene in various cultivars of transgenic plum and establishes that the bul409 promoter is at least equal in effectiveness to the 35SCaMV promoter for gafp-1 expression in transgenic lines of woody plants.

Abstract

The Gastrodia antifungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously generated, transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were created from seeds derived from open pollination of the cultivar Bluebyrd (BB-OP) with gafp-1 under the control of the polyubiquitin promoter bul409 and evaluated for Phytophthora root rot (PRR) and Root knot nematode (RKN) susceptibility. One of nine transgenic lines synthesizing GAFP-1 exhibited increased tolerance to PRR caused by P. cinnamomi. The same line (BB-OP-1) was also significantly more tolerant to RKN infection caused by Meloidogyne incognita. BB-OP-1 was more resistant to PRR and equally resistant to RKN compared with the cultivar Stanley-derived 4J line, which expresses gafp-1 under the control of the 35SCaMV promoter. GAFP-1 synthesis in BB-OP-1 was not elevated by pathogen infection, suggesting that the bul409 promoter is not inducible in the plum/GAFP-1 system. This study confirms the usefulness of the gafp-1 gene in various cultivars of transgenic plum and establishes that the bul409 promoter is at least equal in effectiveness to the 35SCaMV promoter for gafp-1 expression in transgenic lines of woody plants.

South Carolina and Georgia are major peach-producing states in the southern United States. Several soilborne organisms cause significant problems on peach in South Carolina, including PRR and RKN. PRR is a disease with economic impact on the production of peach (P. persica) and other Prunus species worldwide mainly as a result of tree mortality (Haygood et al., 1986; Kephart and Dunegan, 1948; Kouyeas, 1971; Stylianides et al., 1985). In the southeastern United States, the disease is primarily caused by P. cinnamomi (Erwin and Ribeiro, 1996). Excessive soil moisture, moderate temperature, and rootstock susceptibility contribute to disease development (Browne and Mircetich, 1996). The soilborne pathogen is difficult to control even with fungicide treatments. Current chemical control options include the application of fosetyl–aluminum and mefenoxam. Fumigants 1,3-dichloropropen and chloropicrin are also recommended for management of soilborne pathogens (Methyl Bromide Technical Options Committee, 1994). The efficiency of fumigants, however, is dependent on environmental factors such as soil type, soil moisture, and soil temperature (Horton et al., 2010).

Crop damage resulting from plant parasitic nematodes is estimated at ≈$157 billion worldwide annually (Abad et al., 2008). Among the most devastating nematodes are Meloidogyne species. Members of this genus cause root knot disease and have a host range of more than 2000 plant species (Lamberti, 1979; Sasser, 1977). M. incognita, M. javanica, and M. arenaria are the most damaging species in tropical regions (Triantaphyllou, 1985). In the United States, M. incognita is a widespread pathogen of tomatoes, cotton, and soybeans (Castagnone-Sereno, 2006; Ortiz et al., 2010) and one of the most common species reducing fruit production in peach and other stone fruit orchards (Nyczepir et al., 1997). Pre-plant fumigation with 1,3-dichloropropene and metam sodium is still used for RKN control in southeastern peach orchards (Horton et al., 2010), but plants can be killed by 1,3-dichloropropene if planted too soon after fumigation. Control of nematodes is extremely difficult once an orchard is established. An orchard may be in existence for 15 to 25 years.

Creating rootstocks for fruit trees with resistance to fungal root pathogens and nematodes is a desirable component of Integrated Pest Management practices. Classical breeding has in the past been the only method available to develop disease-resistant rootstocks. Progress has been made in the example of the peach rootstocks KID I, PR204, GF305, and GF677, which showed some resistance against Phytophthora cactorum and P. megasperma (Thomidis et al., 2001). Nevertheless, progress has been slow and sources of resistance available for conventional breeding are limited. Genetic engineering offers a complementary method of developing resistance that can greatly expand the pool of resistance genes and offers a way to test these genes in a shorter timeframe.

Genes expressing mannose-binding lectins in monocotyledonous species have been used to generate transgenic plants resistant to a wide range of pathogenic and pest organisms (Peumans and Van Damme, 1995). The Gastrodia anti fungal protein is a mannose-binding lectin with antifungal activity in vitro to Valsa ambiens (Wang et al., 2001) and other fungal pathogens, including Armillaria mellea, Rhizoctonia solani, Gibberella zeae, Ganoderma lucidum, and Botrytis cinerea (Hu and Huang, 1994; Xu et al., 1998). GAFP expression in transgenic cotton increased resistance to Verticillium wilt (Wang et al., 2004) and, in contrast to other lectins, GAFP-1 showed efficacy against RKN and PRR in transgenic line 4J derived from open pollination (OP) of the plum cultivar Stanley (Nagel et al., 2008). The gafp-1 gene in line 4J was under the control of the CaMV35S promoter. Furthermore, line 5D, expressing the gafp-1 gene, resulted in the development of fewer eggs and juveniles of ring nematode, M. xenoplax (Nyczepir et al., 2009). Although GAFP-1 protein expression in plum showed increased resistance to fungal and non-fungal pathogens (Nagel et al., 2008), higher levels of resistance were desired. The promoter bul409 was shown to be more active in transgenic potato compared with the CaMV35S promoter lines and expression level of beta-glucuronidase (GUS) in transgenic potato plants with the polyubiquitin promoter bul409 was found 30-fold higher when compared with lines with the CaMV35S promoter (Rockhold et al., 2008). The objectives of this study were to generate ‘Bluebyrd’ plum lines expressing gafp-1 under the control of the bul409 promoter, to confirm GAFP-1 protein expression in transgenic lines, and to determine their susceptibility to P. cinnamomi and M. incognita relative to the susceptibility of untransformed controls and the previously characterized 4J line.

Materials and Methods

Generation of transgenic plum lines expressing gafp-1 under the control of the bul409 promoter.

The gafp-1 gene, under the control of the bul409 promoter and the Ubi3 terminator, was inserted at the multiple cloning site, with HindIII and SacI, of the pBINPLUS/ARS vector (Fig. 1) (Belknap et al., 2008). Agrobacterium tumefaciens strain EHA 105 (Hood et al., 1993) was transformed with the pBINPLUS/ARS-gafp-1 vector and prepared for infection as described previously (González-Padilla et al., 2003).

Fig. 1.
Fig. 1.

Schematic diagram of pBINPLUS/ARS vector with insertion of the gafp-1 gene placed under bul409 promoter and Ubi3-T (terminator).

Citation: HortScience horts 46, 7; 10.21273/HORTSCI.46.7.975

For plant transformation, ‘Bluebyrd’ plum mature seed hypocotyl slices were used as the source of explants. Transgenic plant production was performed using the method described by Petri et al. (2008). Briefly, endocarps were removed and seeds were soaked in a 1% sodium hypochlorite solution for 30 min followed by rinsing with sterile distilled water. Epicotyle and radicle were removed and the hypocotyl was sliced into three cross-sections. Media for transformation and shoot regeneration was as described by González-Padilla et al. (2003). Explants were cocultured with A. tumefaciens in shoot regeneration medium (SRM) without antibiotics but supplemented with 2,4-D. After 3 d, explants were transferred to SRM supplemented with 80 mg·L−1 kanamycin and 300 mg·L−1 timentin. As shoots appeared, explants were transferred to shoot-growing medium containing 80 mg·L−1 kanamycin and 300 mg·L−1 timentin. When shoots elongated to ≈1 cm, they were separated from the clusters and placed in rooting medium containing 40 mg·L−1 kanamycin and 300 mg·L−1 timentin. Plantlet acclimatization and establishment in a greenhouse was performed also as described by Petri et al. (2008).

Transgenic lines were derived from seeds from OP of ‘Bluebyrd’ plum. ‘Bluebyrd’ plum is self-incompatible; therefore, transgenic lines were half-siblings sharing the same female parent, ‘Bluebyrd’. These lines as well as control lines derived from OP of ‘Bluebyrd’ are referred to in this report as BB-OP. A total of 18 BB-OP transgenic lines, two each of empty vector BB-OP and ‘Stanley’ OP control lines and five each of wild type BB-OP and ‘Stanley’ control lines were used for comparison with the previously characterized 4J plum line included in this study (Nagel et al., 2008). In this line, the gafp-1 gene is driven by the CaMV35S promoter instead of the bul409 promoter.

Determination of gafp-1 gene copy numbers in transgenic plum lines.

DNA was isolated from young and fully expanded leaves of transformed and non-transformed BB-OP lines as described previously (Kobayashi et al., 1998). Briefly, 10 to 15 μg DNA was digested with BamHI (New England Biolabs, Ipswich, MA), separated on a 1% (w/v) agarose gel, and blotted to a positively charged nylon membrane (Roche Diagnostics Corporation, Indianapolis, IN). The membrane was hybridized with a Digoxigenin-11-dUTP alkali-labile (Roche Diagnostics Corporation) -labeled probe coding for gafp-1 cDNA. The probe was generated by polymerase chain reaction using gafp-1-specific primers (Wang et al., 2001).

Detection of Gastrodia antifungal protein-1 protein in transgenic plum lines.

Gastrodia antifungal protein (anticipated size 12 kDa) was detected using immunoblot analysis from root tissue of transformed and non-transformed plum lines from 9-month-old trees as described previously (Nagel et al., 2010). Line 4J (positive control) and a randomly selected ‘Stanley’ empty-vector control line (negative control) were included as references (Nagel et al., 2008). Briefly, total protein was extracted from root tissue of transformed and non-transformed plum plants using TRI reagent® (Sigma-Aldrich, St. Louis, MO). Total protein (20 μg) was used to perform sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 15% Tris-HCl ready Gels (Bio-Rad Laboratories, Hercules, CA). Protein was transferred to an immunoblot polyvinylidene fluoride membrane (Bio-Rad Laboratories), and immunoblotting was performed using rabbit anti-GAFP-1 polyclonal antisera developed by Zymed® Laboratories and goat antirabbit alkaline phosphatase conjugated antibodies (Promega Corp., Madison, WI). Band detection was accomplished using a solution of BCIP/NBT tablet. This experiment was performed twice for each transgenic, empty vector, and wild-type control line. Results were reproducible and were combined. Expression of GAFP-1 was scored visually relative to the expression of GAFP-1 in the 4J line: equal to (+++), up to 50% less (++), and less than 50% (+).

Selection and propagation of transgenic plum lines.

Transgenic lines revealing a strong, consistent GAFP-1 protein signal on immunoblots were used for further experiments. The performance of BB-OP lines was compared with previously characterized line 4J expressing GAFP under the CaMV35S promoter (Nagel et al., 2008). BB-OP-0 and ‘Stanley’ empty vector and untransformed lines were included as negative controls. Vegetative propagation of the plant material was carried out in a biosafety Level 2 greenhouse under constant temperature (27 ± 5 °C) and light conditions (16/8 h day/night). Original transformed lines (T0 lines) were pruned every 4 weeks to stimulate shoot growth. Shoots (15 to 20 cm) were pruned off the T0 lines, gently scraped at the cut end, and dipped into “ROOTECH” Original Cloning Gel (Technaflora Plant Product Ltd., Port Coquitlam, B.C., Canada). Shoots were placed 3 to 4 cm deep in sterile vermiculite in 36-well plastic trays (25 × 50 cm) and covered with a lid to prevent dehydration. Plants were misted and watered daily and fertilized once per week. Fungicide applications (Pristine 0.019% a.i.; BASF, Research Triangle Park, NC) were applied with a mister as needed to control fungal colonization of emerging leaves as a result of the humid condition during propagation.

Disease susceptibility screening.

‘Bluebyrd’ lines BB-OP-1, BB-OP-3, BB-OP-17, BB-OP-18, BB-OP-21, BB-OP-8 EV, non-transformed lines BB-OP-0, BB-OP-30, BB-OP-31, ‘Stanley’ 4J, and ‘Stanley’ untransformed controls were investigated for susceptibility to PRR as described previously (Nagel et al., 2008). P. cinnamomi isolate 05-1127 was obtained from naturally infected peach and had been used for similar studies (Nagel et al., 2008). Briefly, P. cinnamomi was grown on PARP selective medium in the dark at 22 °C for 3 d. Agar plugs (6 mm) with actively growing mycelium were added to 500-mL Erlenmeyer flasks containing 300 mL of a sterile, V8-juice:vermiculite (1:2 v/v) mixture. The flasks were incubated in the dark at 22 °C for 8 weeks.

Experimental plum lines were transplanted to 5-cm diameter plastic torpedo pots containing ≈400 cm3 sterile potting soil mixed with 2% of the infested or non-infested V8-juice:vermiculite. Pots were suspended in plastic racks (10 pots per rack) and placed in 10-gallon plastic bins. For error control, all treatments were arranged in a randomized complete block design. Plants were watered as needed and after 1 week were flooded for 48 h to promote infection. Disease symptoms were evaluated every other day. Shoot symptoms were rated as: 0 = healthy plant, 1 = less than 25% of the plant showing chlorosis and necrosis, 2 = 25% to 50% of the plant showing chlorosis and necrosis, 3 = 50% to 75% of the plant showing chlorosis and necrosis, and 4 = greater than 75% of the plant exhibiting chlorosis and necrosis (Fig. 2). The experiment was concluded after 30 d, when the majority of inoculated control plants had severe wilting. Random root pieces were sampled from inoculated seedlings, surface-sterilized, and plated on PARPH [PARP + 50 mg 5-methylisoxazol- 3-ol (hymexazol)] selective medium (Jeffers, 2006) to confirm the presence of P. cinnamomi. The disease severity score was calculated as described previously (Nagel et al., 2008). The entire experiment was performed twice with three replicates per experiment.

Fig. 2.
Fig. 2.

Disease symptoms of ‘Bluebyrd’ plum lines 30 days after Phytophthora cinnamomi inoculation. Score 0 = asymptomatic plant (not inoculated); score 1 = less than 25% wilted; score 2 = 25% to 50% wilted; score 3 = more than 50% to 75% wilted; score 4 = more than 75% wilted.

Citation: HortScience horts 46, 7; 10.21273/HORTSCI.46.7.975

Three-month-old plants of line BB-OP-1, 4J, ‘Stanley’ untransformed control, and BB-OP-0 untransformed control were used to assess susceptibility to M. incognita as described previously (Nagel et al., 2008). Briefly, plum plants were transplanted into 20-cm diameter plastic pots containing 1000 cm3 of sterile sand:vermiculite (1:1 vol/vol). Root-knot nematode egg inoculum was extracted from infected tomato roots (cv. Rutgers) using a 10% NaOCl solution diluted in tap water. Twelve-week-old plum plants were inoculated with 6000 eggs per pot by creating three holes (≈1 cm deep) in the medium around the base of each plant and pipetting 2 mL (1000 eggs/mL) into each of the three holes. Negative control plants received tap water. For error control, all treatments were arranged in a randomized complete block design. The study was ended 60 d after inoculation and the number of egg masses per root system, number of eggs per root system, and fresh root weight were determined. BB-OP-1 was selected for the RKN assay out of five BB-OP transgenic lines used for the PRR assay based on its low disease severity score in PRR assays compared with other transgenic lines. After weighing the roots, they were stained using a 20% (v/v) solution of McCormick Schilling red food color (Thies et al., 2002) for 25 min, after which the roots were rinsed with tap water and blotted dry. Egg masses were observed under ×20 magnification. The number of galls and egg masses was determined per plant and normalized using root fresh weight to calculate the numbers per gram fresh root weight. M. incognita populations were originally isolated from infected peach in Georgia. The experiment was performed twice with five replicates.

Gastrodia antifungal protein -1 synthesis in roots of transgenic lines before and after inoculation.

To determine whether the bul409 promoter is pathogen-inducible, total protein was extracted as described from roots of BB-OP-1 1 d before and 5 d after inoculation with P. cinnamomi and 1 d before and 30 d after inoculation with M. incognita. Immunoblot analysis was conducted as described previously.

Statistical analysis.

Bartlett's test for homogeneity of variances was performed for repeated experiments. Data sets with homogeneous variances were combined and analyzed for significant differences between each line. In regard to the ordinal Phytophthora disease rating values, a non-parametric comparison using the chi-square test revealed no differences between treatments compared with the means calculated with the parametric method. Thus, for simplicity, we show means and se bars of the parametric analysis. Values were analyzed using the general linear model or analysis of variance and least significant difference mean separation procedures of SAS (Version 9.2; SAS Institute, Cary, NC).

Results

A total of 18 plum lines were obtained from gafp-1 transformation of individual BB-OP seeds and 17 tested positive for the presence of gafp-1. In addition, five non-transformed plants from seeds of ‘Bluebyrd’ (BB-OP-0, BB-OP-30, BB-OP-31, BB-OP-32, and BB-OP-33) and two transgenic empty vector control lines (BB-OP-7 EV and BB-OP-8 EV) were included in this study (Table 1). BB-OP lines were not phenotypically different from each other or from the non-transformed or empty vector control lines based on visual assessment of shoot length, leaf shape, size, and color (data not shown).

Table 1.

Number of gafp-1 copies and GAFP-1 protein expression in roots of plum lines used in this study.

Table 1.

Southern hybridization showed a range of copy numbers from zero to five inserts (Table 1). Only nine of the 18 BB-OP lines revealed a GAFP-1 signal. The consistently strongest signals (data from at least two independent experiments) were found for lines BB-OP-1, -3, -17, -18, and -21 (Table 1; Fig. 3A–B). No GAFP-1 signal was detected in either the empty vector controls or the non-transformed control lines. Based on more than four different immunoblot assays, the GAFP-1 signal strength of BB-OP-3, which exhibited consistently one of the highest GAFP-1 signals, was comparable in intensity to line 4J.

Fig. 3.
Fig. 3.

Immunoblot analysis of total protein extracts (20 μg) from root tissue; (A) Lane 1: purified GAFP-1 (≈12 kDa); Lanes 2 and 4: nontransformed ‘Stanley’ control and BB-OP, respectively; Lanes 3 and 5: transgenic 4J (‘Stanley’ plum seedling) and BB-OP-3 (‘Bluebyrd’ plum seedling) lines, respectively. (B) Lane 1: GAFP-1 (≈12 kDa); Lane 2: 4J and Lanes 3 to 8: transgenic ‘Bluebyrd’ plum lines BB-OP-1, BB-OP-2, BB-OP-3, BB-OP-17, BB-OP-18, and BB-OP-21, respectively. GAFP-1 = Gastrodia antifungal protein; BB-OP = open-pollinated ‘Bluebyrd’.

Citation: HortScience horts 46, 7; 10.21273/HORTSCI.46.7.975

The five lines with the highest GAFP-1 signals (BB-OP-1, -3, -17, -18, and -21) were selected for PRR disease tests based on their GAFP-1 expression. Included were also one empty vector line (BB-OP-8 EV) and several untransformed control lines BB-OP-0, BB-OP-30, and BB-OP-31 to sample the natural half-sibling genetic variation in disease susceptibility. The results of two independent experiments were not significantly different (P = 0.3044, α = 0.05) and the data sets were combined. BB-OP-1 was significantly more resistant to PRR disease compared with other BB-OP lines and compared with the control lines (Fig. 4). None of the other transgenic BB-OP lines were statistically different from the BB-OP control lines. In addition, disease severity of line 4J was numerically but not significantly different from the ‘Stanley’ control line (P = 0.3088, at α = 0.05; Fig. 4). BB-OP-3 showed strong GAFP expression, comparable to 4J expression, and demonstrated similar resistance in PRR disease tests. BB-OP-1 was statistically better for PRR disease control compared with 4J at the α = 0.1 level (data not shown).

Fig. 4.
Fig. 4.

Disease severity of 3-month-old plum lines BB-OP-1, BB-OP-3, BB-OP-17, BB-OP-18, BB-OP-21, BB-OP-8 EV, ‘Bluebyrd’ control (mixture of lines BB-OP-0, BB-OP-30, and BB-OP- 31), 4J, and ‘Stanley’ control 30 d after inoculation with Phytophthora cinnamomi. Bars represent the average of two experiments with three replicates each. Bars with the same letter are not significantly different (α = 0.05). BB-OP = open-pollinated ‘Bluebyrd’.

Citation: HortScience horts 46, 7; 10.21273/HORTSCI.46.7.975

Based on the PRR test results, the best performing BB-OP line (BB-OP-1) was subjected to RKN disease screening tests and compared with BB-OP-0 untransformed control, ‘Stanley’ untransformed control, and line 4J. BB-OP-0 was chosen as the sole control line because it represented an average level of susceptibility to PRR among BB-OP control lines. Between the independent experiments, no statistical differences were found (P = 0.6687 for eggs/g of root; P = 0.5145 for egg mass/g of root; and P = 0.6154 at α = 0.05) for galls/g of root; thus, the combined data set is shown. For all parameters tested (eggs/g of root, egg mass/g of root, and galls/g of root), line BB-OP-1 as well as 4J performed significantly better than the corresponding controls (Fig. 5). No statistical differences were found between the two transgenic lines 4J and BB-OP-1 (P = 0.2782 for eggs/g of root; P = 0.8221 for egg mass/g of root; P = 0.3377 at α = 0.05 for galls/g of root; Fig. 5).

Fig. 5.
Fig. 5.

Reproduction of Meloidogyne incognita on roots of ‘Stanley’ control line, ‘Bluebyrd’ control line (BB-OP-0), transgenic lines 4J, and BB-OP-1. (A) Number of eggs per gram of root; (B) egg mass (egg mass per gram of fresh root); and (C) gall formation (galls per gram of root). Shown is the combined data set of two independent experiments. Bars represent the average of two experiments with five replicates each. Bars with the same letter are not significantly different (α = 0.05). BB-OP = open-pollinated ‘Bluebyrd’.

Citation: HortScience horts 46, 7; 10.21273/HORTSCI.46.7.975

The inducibility of gafp-1 after pathogen exposure was estimated using immunoblot analysis. The signals for GAFP-1 in immunoblot analyses were not noticeably higher in root tissue of line BB-OP-1 5 d after inoculation with P. cinnamomi (Fig. 6A) compared with the non-inoculated BB-OP-1 root tissue. Likewise, no elevated GAFP-1 signal was detected in root tissue 30 d after inoculation with M. incognita (Fig. 6B) compared with the non-inoculated root tissue.

Fig. 6.
Fig. 6.

Immunoblot analysis showing GAFP-1 in 20 μg of total protein from root tissue of transgenic line BB-OP-1 (A) before (Lane 2) and 5 d after (Lane 3) inoculation with Phytophthora cinnamomi and (B) before (Lane 2) and 30 d after (Lane 3) inoculation with Meloidogyne incognita. GAFP-1 = Gastrodia antifungal protein; BB-OP = open-pollinated ‘Bluebyrd’.

Citation: HortScience horts 46, 7; 10.21273/HORTSCI.46.7.975

Discussion

Among 17 BB-OP lines that tested positive for the presence of gafp-1 DNA, only nine were found to express the GAFP-1 protein in immunoblot studies. The failure of some transgenic plant lines to produce heterologous protein despite successful insertion of the target gene has been described before in other systems, including transgenic potato using the potato leafroll virus replicase transgene (Ehrenfeld et al., 2004) and transgenic walnut using the crylA(c) gene (Dandekar et al., 1998). In addition, variation in the level of transgene expression is common among transformed plants. It is not completely understood why inserted transgenes do not function in some transgenic plants, but it is possible that the insertion of the transgene into the plant genome occurs at locations that do not support gene expression. Kumar and Fladung (2001) showed that AT-rich regions in the genome of transgenic aspen (Populus) may be involved in the defense against foreign gene insertions. In studies of transgenic tobacco and tomato, ATTTA sequences and A+T-rich regions affected the protein expression level in plants (Perlak et al., 1991). Another possible explanation is that mutations in the promoter or the gafp-1 gene occurred during transformation.

Line BB-OP-1 showed significantly less severe disease symptoms in PRR and RKN tests, although it did not exhibit the highest GAFP-1 synthesis level among transgenic lines. Although BB-OP-3 had a greater number of copies of the gafp-1 gene compared with BB-OP-1 and expressed higher levels of GAFP, it was more susceptible than BB-OP-1 to P. cinnamomi. Similarly, transgenic A. thaliana lines with high levels of disease resistance did not correspond to the ones with the highest expression of the insecticidal lectin GNA (Galanthus nivalis agglutinin) in roots (Ripoll et al., 2003) Expression of GAFP-1 in transgenic plum line 5D was higher than that of lines 4J and 4I, but 5D was more susceptible in PRR and RKN disease tests (Nagel et al., 2008). It is possible that the multiple insertions of gafp-1 copies in many of the BB-OP lines had an effect on the physiology of the plant that may have impaired inherent disease resistance, counteracting the antimicrobial effect of elevated GAFP-1 levels. In immunoblot analyses, proteins with higher molecular weight compared with GAFP-1 (12 kDa) reacted with our GAFP-1 probe. The additional bands were observed in both transgenic and control plants and likely signaled nonspecific binding of the polyclonal antibody. Similar nonspecific binding has been observed in previous studies on tobacco (Cox et al., 2006) and plum (Nagel et al., 2008). Nonspecific binding of a polyclonal antibody is not uncommon, as shown for apple shoot-extracted Vfa1 and Vfa2 proteins (Malnoy et al., 2008).

BB-OP-1 displayed resistance against RKN, but its performance in the PRR experiment was not superior to the ‘Stanley’-derived 4J line. Both lines had reduced numbers of galls, egg masses, and eggs compared with inoculated control lines, effects that had previously been noted for the 4J line (Nagel et al., 2008). The gafp-1-expressing lines except BB-OP-1 and BB-OP-3 under the control of bul409 did not result in less disease when compared with the lines with the CaMV35S promoter. Polyubiquitin promoters such as the bul409 have shown enhanced expression of the reporter gene (GUS) in various transgenic plants such as transgenic potato (Rockhold et al., 2008) and rice (Lu et al., 2008). The performance of the bul409 promoter may be dependent on the host plant. For example, the polyubiquitin promoter GUBQ1 did not elevate the expression of the GUS reporter gene compared with the CaMV35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia (Joung and Kamo, 2006). In transgenic wheat, the expression of the insecticidal lectin GNA under control of an ubiquitin promoter was significantly lower compared with its expression in transgenic rice (Stoeger et al., 1999).

The bul409 promoter had been shown to be wound-inducible in transgenic potato lines (Rockhold et al., 2008). Expression of bul409 promoter-driven GUS mRNA levels was higher in wounded tubers and leaves (Rockhold et al., 2008). Inducibility was not demonstrated in our study. Neither inoculation with P. cinnamomi nor inoculation with M. incognita increased GAFP-1 synthesis in transgenic plums suggesting that there was no inducibility in gafp-1 gene expression. The two studies, however, cannot be directly compared because the mRNA level, but not protein expression, was measured in potato tubers and leaves, whereas only the protein production was measured in this study. It is possible that no increase of GUS protein levels occurred in potatoes despite the increase of mRNA levels. It is unlikely that the inducibility of bul409 may occur in wounded but not in pathogen-induced tissue because wounding and pathogen responses share a number of components in their signaling pathways (Maleck and Dietrich, 1999).

This study established that the gafp-1 gene is stable in transgenic plum lines. Line 4J was developed from ‘Stanley’ seed (Nagel et al., 2008) and has since been grown in the greenhouse under conditions allowing continuous, vegetative growth. After 4 years, GAFP-1 synthesis and pathogen resistance were consistent with that previously described (Nagel et al., 2008) with the exception that resistance to PRR was only increased over controls numerically but was not statistically significant. In the present study, fewer replicates were used compared with the earlier study. Stability of GAFP-1 synthesis was also confirmed in transgenic tobacco lines, which were generated in 2004 (Cox et al., 2006) and had been used for GAFP-1 isolation continuously until 2010 (Nagel et al., 2010). The present study confirms the potential for gafp-1 as a disease resistance gene in woody plants. Increased disease resistance has now been demonstrated in two different sources of plum germplasm, ‘Stanley’ and ‘Bluebyrd’. The long-term stability of GAFP-1 synthesis in transgenic plum lines was also confirmed. The suitability of ubiquitin promoter bul409 for gafp-1 expression plum was established and for BB-OP-1, a relatively high level of resistance to PRR (at α = 0.1, data not shown) was observed that was comparable to the level achieved in transgenic line 4J in which the gafp-1 gene was under the 35SCaMV promoter. In conclusion, this study confirms the usefulness of the gafp-1 gene in various cultivars of transgenic plum and establishes that the bul409 promoter is at least equal in effectiveness to the 35SCaMV promoter for gafp-1 expression in transgenic lines of woody plants.

Literature Cited

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    • Search Google Scholar
    • Export Citation
  • Belknap, W., Rockhold, D. & McCue, K. 2008 pBINPLUS/ARS: An improved plant transformation vector based on pBINPLUS Biotechniques 44 753 756

  • Browne, G.T. & Mircetich, S.M. 1996 Effects of month of inoculation on severity of disease caused by Phytophthora species in apple root, crowns and excised shoots Phytopathology 86 290 294

    • Search Google Scholar
    • Export Citation
  • Castagnone-Sereno, P. 2006 Genetic variability and adaptive evolution in parthenogenetic root-knot nematodes Heredity 96 282 289

  • Cox, K., Layne, D., Scorza, R. & Schnabel, G. 2006 Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco Planta 224 1373 1383

    • Search Google Scholar
    • Export Citation
  • Dandekar, M., McGranahan, G., Vail, P., Uratsu, S., Leslie, C. & Tebbets, J. 1998 High levels of expression of full-length cry1A(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos Plant Sci. 131 181 193

    • Search Google Scholar
    • Export Citation
  • Ehrenfeld, N., Romano, E., Serrano, C. & Arce-Johnson, P. 2004 Replicase mediated resistance against potato leafroll virus in potato Desirée plants Biol. Res. 37 71 82

    • Search Google Scholar
    • Export Citation
  • Erwin, D. & Ribeiro, O. 1996 Isolation and detection of Phytophthora 8 41 Erwin D. & Ribeiro O. Phytophthora disease world wide APS Press St. Paul, MN

  • González-Padilla, M., Webb, K. & Scorza, R. 2003 Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (Prunus domestica L.) Plant Cell Rep. 22 38 45

    • Search Google Scholar
    • Export Citation
  • Haygood, R.A., Graves, C.H. & Ridings, W.H. 1986 Phytophthora root rot and stem canker of peach trees in Mississippi Plant Dis. 70 866 868

  • Hood, E., Gelvin, S., Melchers, L. & Hoekema, A. 1993 New Agrobacterium helper plasmids for gene transfer to plants Transgenic Res. 2 208 218

  • Horton, D., Brannen, P., Bellinger, B. & Ritchie, D. 2010 Southeastern peach, nectarine and plum pest management and culture guide Univ. of Georgia Coop. Ext. Serv. Bull. #1171. 17 Oct. 2010. <http://www.ent.uga.edu/peach/PeachGuide.pdf>.

    • Search Google Scholar
    • Export Citation
  • Hu, Z. & Huang, Q.Z. 1994 Induction and accumulation of the antifungal protein in Gastrodia elata Acta Bot. Yunnanica 16 169 177

  • Jeffers, S. 2006 Identifying species of Phytophthora Clemson University 17 Nov. 2010 <http://fhm.fs.fed.us/sp/sod/misc/culturing_species_phytophthora.pdf>.

    • Search Google Scholar
    • Export Citation
  • Joung, Y. & Kamo, K. 2006 Expression of a polyubiquitin promoter isolated from Gladiolus Plant Cell Rep. 25 1081 1088

  • Kephart, J.E. & Dunegan, J.C. 1948 Infection of seedling peach stems by zoospores of Phytophthora cactorum Phytopathology 38 580 581

  • Kobayashi, N., Horikoshi, T., Katsuyama, H., Handa, T. & Takayanagi, K. 1998 A simple and efficient DNA extraction method for plants, especially woody plants Plant Tissue Cult. Biotechnol. 4 76 80

    • Search Google Scholar
    • Export Citation
  • Kouyeas, H. 1971 On the apoplexy of fruit trees caused by Phytophthora species Ann. Inst. Phytopath. Benaki N.S. 10 163 170

  • Kumar, S. & Fladung, M. 2001 Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen Planta 213 731 740

    • Search Google Scholar
    • Export Citation
  • Lamberti, F. 1979 Economic importance of Meloidogyne species in subtropical and mediterranean climates 342 357 Lamberti F. & Taylor C.E. Root-knot nematodes (Meloidogyne species) Systematic, biology and control. Academic Press New York, NY

    • Search Google Scholar
    • Export Citation
  • Lu, J., Sivamani, E., Li, X. & Qu, R. 2008 Activity of the 5′ regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analyzed by both GUS and GFP reporter genes Plant Cell Rep. 27 1587 1600

    • Search Google Scholar
    • Export Citation
  • Maleck, K. & Dietrich, R. 1999 Defense on multiple fronts: How do plants cope with diverse enemies? Trends Plant Sci. 4 215 219

  • Malnoy, M., Xu, M., Borejsza-Wysocka, E., Korban, S. & Aldwinckle, H. 2008 Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease Mol. Plant Microbe Interact. 21 448 458

    • Search Google Scholar
    • Export Citation
  • Methyl Bromide Technical Options Committee 1994 Montreal protocol on substances that deplete the ozone layer 19 Oct. 2010 <http://www.unep.org/ozone/teap/Reports/MBTOC/MBTOC94.pdf>.

    • Search Google Scholar
    • Export Citation
  • Nagel, A., Kalariya, H. & Schnabel, G. 2010 The gastrodia antifungal protein (GAFP-1) and its transcript are absent from scions of chimeric-grafted plum HortScience 45 188 192

    • Search Google Scholar
    • Export Citation
  • Nagel, A.K., Scorza, R., Petri, C. & Schnabel, G. 2008 Generation and characterization of transgenic plum lines expressing the gastrodia-anti fungal protein HortScience 43 1514 1521

    • Search Google Scholar
    • Export Citation
  • Nyczepir, A., Nagel, A.K. & Schnabel, G. 2009 Host status of three transgenic plum lines to Mesocriconema xenoplax HortScience 44 1932 1935

  • Nyczepir, A.P., Miller, R.W. & Beckman, T.G. 1997 Root-knot nematodes on peach in the southeastern United States: An update and advances Afr. Plant Prot. 3 115

    • Search Google Scholar
    • Export Citation
  • Ortiz, B.V., Perry, C., Goovaerts, P., Vellidis, G. & Sullivan, D. 2010 Geostatistical modeling of the spatial variability and risk areas of southern root-knot nematodes in relation to soil properties Geoderma 156 243 252

    • Search Google Scholar
    • Export Citation
  • Perlak, F., Fuches, R., Dean, D., McPhersont, S. & Fischhoff, D. 1991 Modification of the coding sequence enhances plant expression of insect control protein genes Proc. Natl. Acad. Sci. USA 88 3324 3328

    • Search Google Scholar
    • Export Citation
  • Petri, C., Webb, K., Hily, J.M., Dardick, C. & Scorza, R. 2008 High transformation efficiency in plum (Prunus domestica L.): A new tool for functional genomics studies in Prunus species Mol. Breed. 22 581 591

    • Search Google Scholar
    • Export Citation
  • Peumans, W.J. & Van Damme, E.J.M. 1995 Lectins as plant defense proteins Plant Physiol. 109 347 352

  • Ripoll, C., Favery, B., Lecomte, P., Van Damme, E., Peumans, W., Abad, P. & Jouanin, L. 2003 Evaluation of the ability of lectin from snowdrop (Galanthus nivalis) to protect plants against root-knot nematodes Plant Sci. 164 517 523

    • Search Google Scholar
    • Export Citation
  • Rockhold, D., Chang, S., Taylor, N., Allen, P.V., McCue, K. & Belknap, W. 2008 Structure of two Solanum bulbocastanum polyubiquitin genes and expression of their promoters in transgenic potatoes Amer. J. Potato Res. 85 219 226

    • Search Google Scholar
    • Export Citation
  • Sasser, J.N. 1977 Worldwide dissemination and importance of the root-knot nematodes Meloidogyne species J. Nematol. 22 585 589

  • Stoeger, E., Williams, S., Christou, P., Down, R. & Gatehouse, J. 1999 Expression of the insecticidal lectin from the snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: Effect on predation by the grain aphid Sitobion avenae Mol. Breed. 5 65 73

    • Search Google Scholar
    • Export Citation
  • Stylianides, D.C., Chitzanidis, A. & Theochari-Athanasiou, I. 1985 Evaluation of resistance to Phytophthora species Rhizoctonia solani in stone fruit rootstocks Options Mediterraneennes, CIHEAM. 85 73 78

    • Search Google Scholar
    • Export Citation
  • Thies, J., Merrill, S. & Corley, E. 2002 Red food coloring stain: New, safer procedures for staining nematodes in roots and egg masses on root surfaces J. Nematol. 34 179 181

    • Search Google Scholar
    • Export Citation
  • Thomidis, T., Cullum, J., Elena, K. & Jeffers, S. 2001 Relative resistance of four peach rootstocks to Phytophthora cactorum and P. megasperma J. Phytopathol. 149 599 604

    • Search Google Scholar
    • Export Citation
  • Triantaphyllou, A.C. 1985 Cytogenetics, cytotaxonomy and phylogeny of root-knot nematodes 113 126 Sasser J.N. & Carter C.C. An advanced treatise on Meloidogyne North Carolina State University Graphics Raleigh, NC

    • Search Google Scholar
    • Export Citation
  • Wang, X., Bauw, G., Van Damme, E., Peumans, W., Chen, Z., Van Montagu, M., Angenon, G. & Dillen, W. 2001 Gastrodianin-like mannose-binding proteins: A novel class of plant proteins with antifungal properties Plant J. 25 651 661

    • Search Google Scholar
    • Export Citation
  • Wang, Y., Chen, D., Wang, D., Huang, Q., Yao, Z., Liu, F., Wei, X., Li, R., Zhang, Z. & Sunday, Y. 2004 Over-expression of Gastrodia antifungal protein enhances Verticillium wilt resistance in coloured cotton Plant Breed. 123 454 459

    • Search Google Scholar
    • Export Citation
  • Xu, Q., Liu, Y., Wang, X., Gu, H. & Chen, Z. 1998 Purification and characterization of a novel anti-fungal protein from Gastrodia elata Plant Physiol. Biochem. 36 899 905

    • Search Google Scholar
    • Export Citation

Contributor Notes

This study was funded in part by USDA-CSREES S-RIPM grant no. 2005-34103-15588 and USDA-CSREES special grant no. 2004-34126-14388 under project number SC-1000642 as well as USDA NRI grant no. 2002-35319-12527 and the South Carolina Peach Council.

Technical contribution number 5892 of the Clemson University Experiment Station.

We thank P. Karen Bryson for technical assistance, Dr. Steven Jeffers for supplying the Phytopththora strain, Dr. Patrick D. Gerard for statistical advice, and Dr. Alexis Nagel for helpful comments on this manuscript during the preparation period.

To whom reprint requests should be addressed; e-mail schnabe@clemson.edu.

  • View in gallery

    Schematic diagram of pBINPLUS/ARS vector with insertion of the gafp-1 gene placed under bul409 promoter and Ubi3-T (terminator).

  • View in gallery

    Disease symptoms of ‘Bluebyrd’ plum lines 30 days after Phytophthora cinnamomi inoculation. Score 0 = asymptomatic plant (not inoculated); score 1 = less than 25% wilted; score 2 = 25% to 50% wilted; score 3 = more than 50% to 75% wilted; score 4 = more than 75% wilted.

  • View in gallery

    Immunoblot analysis of total protein extracts (20 μg) from root tissue; (A) Lane 1: purified GAFP-1 (≈12 kDa); Lanes 2 and 4: nontransformed ‘Stanley’ control and BB-OP, respectively; Lanes 3 and 5: transgenic 4J (‘Stanley’ plum seedling) and BB-OP-3 (‘Bluebyrd’ plum seedling) lines, respectively. (B) Lane 1: GAFP-1 (≈12 kDa); Lane 2: 4J and Lanes 3 to 8: transgenic ‘Bluebyrd’ plum lines BB-OP-1, BB-OP-2, BB-OP-3, BB-OP-17, BB-OP-18, and BB-OP-21, respectively. GAFP-1 = Gastrodia antifungal protein; BB-OP = open-pollinated ‘Bluebyrd’.

  • View in gallery

    Disease severity of 3-month-old plum lines BB-OP-1, BB-OP-3, BB-OP-17, BB-OP-18, BB-OP-21, BB-OP-8 EV, ‘Bluebyrd’ control (mixture of lines BB-OP-0, BB-OP-30, and BB-OP- 31), 4J, and ‘Stanley’ control 30 d after inoculation with Phytophthora cinnamomi. Bars represent the average of two experiments with three replicates each. Bars with the same letter are not significantly different (α = 0.05). BB-OP = open-pollinated ‘Bluebyrd’.

  • View in gallery

    Reproduction of Meloidogyne incognita on roots of ‘Stanley’ control line, ‘Bluebyrd’ control line (BB-OP-0), transgenic lines 4J, and BB-OP-1. (A) Number of eggs per gram of root; (B) egg mass (egg mass per gram of fresh root); and (C) gall formation (galls per gram of root). Shown is the combined data set of two independent experiments. Bars represent the average of two experiments with five replicates each. Bars with the same letter are not significantly different (α = 0.05). BB-OP = open-pollinated ‘Bluebyrd’.

  • View in gallery

    Immunoblot analysis showing GAFP-1 in 20 μg of total protein from root tissue of transgenic line BB-OP-1 (A) before (Lane 2) and 5 d after (Lane 3) inoculation with Phytophthora cinnamomi and (B) before (Lane 2) and 30 d after (Lane 3) inoculation with Meloidogyne incognita. GAFP-1 = Gastrodia antifungal protein; BB-OP = open-pollinated ‘Bluebyrd’.

  • Abad, P., Gouzy, J., Aury, J.M., Castagnone-Sereno, P., Danchin, E.G., Deleury, E., Perfus-Barbeoch, L., Anthouard, V., Artiguenave, F., Blok, V.C., Caillaud, M.C., Coutinho, P.M., Dasilva, C., De Luca, F., Deau, F., Esquibet, M., Flutre, T., Goldstone, J.V., Hamamouch, N., Hewezi, T., Jaillon, O., Jubin, C., Leonetti, P., Magliano, M., Maier, T.R., Markov, G.V., McVeigh, P., Pesole, G., Poulain, J., Robinson-Rechavi, M., Sallet, E., Segurens, B., Steinbach, D., Tytgat, T., Ugarte, E., van Ghelder, C., Veronico, P., Baum, T.J., Blaxter, M., Bleve-Zacheo, T., Davis, E.L., Ewbank, J.J., Favery, B., Grenier, E., Henrissat, B., Jones, J.T., Laudet, V., Maule, A.G., Quesneville, H., Rosso, M.N., Schiex, T., Smant, G., Weissenbach, J. & Wincker, P. 2008 Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita Nat. Biotechnol. 26 909 915

    • Search Google Scholar
    • Export Citation
  • Belknap, W., Rockhold, D. & McCue, K. 2008 pBINPLUS/ARS: An improved plant transformation vector based on pBINPLUS Biotechniques 44 753 756

  • Browne, G.T. & Mircetich, S.M. 1996 Effects of month of inoculation on severity of disease caused by Phytophthora species in apple root, crowns and excised shoots Phytopathology 86 290 294

    • Search Google Scholar
    • Export Citation
  • Castagnone-Sereno, P. 2006 Genetic variability and adaptive evolution in parthenogenetic root-knot nematodes Heredity 96 282 289

  • Cox, K., Layne, D., Scorza, R. & Schnabel, G. 2006 Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco Planta 224 1373 1383

    • Search Google Scholar
    • Export Citation
  • Dandekar, M., McGranahan, G., Vail, P., Uratsu, S., Leslie, C. & Tebbets, J. 1998 High levels of expression of full-length cry1A(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos Plant Sci. 131 181 193

    • Search Google Scholar
    • Export Citation
  • Ehrenfeld, N., Romano, E., Serrano, C. & Arce-Johnson, P. 2004 Replicase mediated resistance against potato leafroll virus in potato Desirée plants Biol. Res. 37 71 82

    • Search Google Scholar
    • Export Citation
  • Erwin, D. & Ribeiro, O. 1996 Isolation and detection of Phytophthora 8 41 Erwin D. & Ribeiro O. Phytophthora disease world wide APS Press St. Paul, MN

  • González-Padilla, M., Webb, K. & Scorza, R. 2003 Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (Prunus domestica L.) Plant Cell Rep. 22 38 45

    • Search Google Scholar
    • Export Citation
  • Haygood, R.A., Graves, C.H. & Ridings, W.H. 1986 Phytophthora root rot and stem canker of peach trees in Mississippi Plant Dis. 70 866 868

  • Hood, E., Gelvin, S., Melchers, L. & Hoekema, A. 1993 New Agrobacterium helper plasmids for gene transfer to plants Transgenic Res. 2 208 218

  • Horton, D., Brannen, P., Bellinger, B. & Ritchie, D. 2010 Southeastern peach, nectarine and plum pest management and culture guide Univ. of Georgia Coop. Ext. Serv. Bull. #1171. 17 Oct. 2010. <http://www.ent.uga.edu/peach/PeachGuide.pdf>.

    • Search Google Scholar
    • Export Citation
  • Hu, Z. & Huang, Q.Z. 1994 Induction and accumulation of the antifungal protein in Gastrodia elata Acta Bot. Yunnanica 16 169 177

  • Jeffers, S. 2006 Identifying species of Phytophthora Clemson University 17 Nov. 2010 <http://fhm.fs.fed.us/sp/sod/misc/culturing_species_phytophthora.pdf>.

    • Search Google Scholar
    • Export Citation
  • Joung, Y. & Kamo, K. 2006 Expression of a polyubiquitin promoter isolated from Gladiolus Plant Cell Rep. 25 1081 1088

  • Kephart, J.E. & Dunegan, J.C. 1948 Infection of seedling peach stems by zoospores of Phytophthora cactorum Phytopathology 38 580 581

  • Kobayashi, N., Horikoshi, T., Katsuyama, H., Handa, T. & Takayanagi, K. 1998 A simple and efficient DNA extraction method for plants, especially woody plants Plant Tissue Cult. Biotechnol. 4 76 80

    • Search Google Scholar
    • Export Citation
  • Kouyeas, H. 1971 On the apoplexy of fruit trees caused by Phytophthora species Ann. Inst. Phytopath. Benaki N.S. 10 163 170

  • Kumar, S. & Fladung, M. 2001 Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen Planta 213 731 740

    • Search Google Scholar
    • Export Citation
  • Lamberti, F. 1979 Economic importance of Meloidogyne species in subtropical and mediterranean climates 342 357 Lamberti F. & Taylor C.E. Root-knot nematodes (Meloidogyne species) Systematic, biology and control. Academic Press New York, NY

    • Search Google Scholar
    • Export Citation
  • Lu, J., Sivamani, E., Li, X. & Qu, R. 2008 Activity of the 5′ regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analyzed by both GUS and GFP reporter genes Plant Cell Rep. 27 1587 1600

    • Search Google Scholar
    • Export Citation
  • Maleck, K. & Dietrich, R. 1999 Defense on multiple fronts: How do plants cope with diverse enemies? Trends Plant Sci. 4 215 219

  • Malnoy, M., Xu, M., Borejsza-Wysocka, E., Korban, S. & Aldwinckle, H. 2008 Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease Mol. Plant Microbe Interact. 21 448 458

    • Search Google Scholar
    • Export Citation
  • Methyl Bromide Technical Options Committee 1994 Montreal protocol on substances that deplete the ozone layer 19 Oct. 2010 <http://www.unep.org/ozone/teap/Reports/MBTOC/MBTOC94.pdf>.

    • Search Google Scholar
    • Export Citation
  • Nagel, A., Kalariya, H. & Schnabel, G. 2010 The gastrodia antifungal protein (GAFP-1) and its transcript are absent from scions of chimeric-grafted plum HortScience 45 188 192

    • Search Google Scholar
    • Export Citation
  • Nagel, A.K., Scorza, R., Petri, C. & Schnabel, G. 2008 Generation and characterization of transgenic plum lines expressing the gastrodia-anti fungal protein HortScience 43 1514 1521

    • Search Google Scholar
    • Export Citation
  • Nyczepir, A., Nagel, A.K. & Schnabel, G. 2009 Host status of three transgenic plum lines to Mesocriconema xenoplax HortScience 44 1932 1935

  • Nyczepir, A.P., Miller, R.W. & Beckman, T.G. 1997 Root-knot nematodes on peach in the southeastern United States: An update and advances Afr. Plant Prot. 3 115

    • Search Google Scholar
    • Export Citation
  • Ortiz, B.V., Perry, C., Goovaerts, P., Vellidis, G. & Sullivan, D. 2010 Geostatistical modeling of the spatial variability and risk areas of southern root-knot nematodes in relation to soil properties Geoderma 156 243 252

    • Search Google Scholar
    • Export Citation
  • Perlak, F., Fuches, R., Dean, D., McPhersont, S. & Fischhoff, D. 1991 Modification of the coding sequence enhances plant expression of insect control protein genes Proc. Natl. Acad. Sci. USA 88 3324 3328

    • Search Google Scholar
    • Export Citation
  • Petri, C., Webb, K., Hily, J.M., Dardick, C. & Scorza, R. 2008 High transformation efficiency in plum (Prunus domestica L.): A new tool for functional genomics studies in Prunus species Mol. Breed. 22 581 591

    • Search Google Scholar
    • Export Citation
  • Peumans, W.J. & Van Damme, E.J.M. 1995 Lectins as plant defense proteins Plant Physiol. 109 347 352

  • Ripoll, C., Favery, B., Lecomte, P., Van Damme, E., Peumans, W., Abad, P. & Jouanin, L. 2003 Evaluation of the ability of lectin from snowdrop (Galanthus nivalis) to protect plants against root-knot nematodes Plant Sci. 164 517 523

    • Search Google Scholar
    • Export Citation
  • Rockhold, D., Chang, S., Taylor, N., Allen, P.V., McCue, K. & Belknap, W. 2008 Structure of two Solanum bulbocastanum polyubiquitin genes and expression of their promoters in transgenic potatoes Amer. J. Potato Res. 85 219 226

    • Search Google Scholar
    • Export Citation
  • Sasser, J.N. 1977 Worldwide dissemination and importance of the root-knot nematodes Meloidogyne species J. Nematol. 22 585 589

  • Stoeger, E., Williams, S., Christou, P., Down, R. & Gatehouse, J. 1999 Expression of the insecticidal lectin from the snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: Effect on predation by the grain aphid Sitobion avenae Mol. Breed. 5 65 73

    • Search Google Scholar
    • Export Citation
  • Stylianides, D.C., Chitzanidis, A. & Theochari-Athanasiou, I. 1985 Evaluation of resistance to Phytophthora species Rhizoctonia solani in stone fruit rootstocks Options Mediterraneennes, CIHEAM. 85 73 78

    • Search Google Scholar
    • Export Citation
  • Thies, J., Merrill, S. & Corley, E. 2002 Red food coloring stain: New, safer procedures for staining nematodes in roots and egg masses on root surfaces J. Nematol. 34 179 181

    • Search Google Scholar
    • Export Citation
  • Thomidis, T., Cullum, J., Elena, K. & Jeffers, S. 2001 Relative resistance of four peach rootstocks to Phytophthora cactorum and P. megasperma J. Phytopathol. 149 599 604

    • Search Google Scholar
    • Export Citation
  • Triantaphyllou, A.C. 1985 Cytogenetics, cytotaxonomy and phylogeny of root-knot nematodes 113 126 Sasser J.N. & Carter C.C. An advanced treatise on Meloidogyne North Carolina State University Graphics Raleigh, NC

    • Search Google Scholar
    • Export Citation
  • Wang, X., Bauw, G., Van Damme, E., Peumans, W., Chen, Z., Van Montagu, M., Angenon, G. & Dillen, W. 2001 Gastrodianin-like mannose-binding proteins: A novel class of plant proteins with antifungal properties Plant J. 25 651 661

    • Search Google Scholar
    • Export Citation
  • Wang, Y., Chen, D., Wang, D., Huang, Q., Yao, Z., Liu, F., Wei, X., Li, R., Zhang, Z. & Sunday, Y. 2004 Over-expression of Gastrodia antifungal protein enhances Verticillium wilt resistance in coloured cotton Plant Breed. 123 454 459

    • Search Google Scholar
    • Export Citation
  • Xu, Q., Liu, Y., Wang, X., Gu, H. & Chen, Z. 1998 Purification and characterization of a novel anti-fungal protein from Gastrodia elata Plant Physiol. Biochem. 36 899 905

    • Search Google Scholar
    • Export Citation
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