Watermelon plants are susceptible to Gummy stem blight disease that considerably reduces yields worldwide. In order to develop non-specific resistance, watermelon cv. Crimson Sweet was transformed with copper inducible isopentenyl transferase (ipt), the rate-limiting step in cytokinin biosynthesis, gene via Agrobacterium tumafaciences (LBA4404). Transformed (ipt) and nontransformed plants were grown at approximately 28-30 °C day, 20-22 °C night and 16 hours daylight under greenhouse conditions. Once the plants initiated new growth both transgenic plants and wild type plants were sprayed with one of three different concentrations (0, 10 & 50 μm) of CuSO4. Plants were sprayed twice to run-off in a twenty-four hour time period before inoculation with the pathogen. Cultures of the pathogen Didymela bryonia (W353) were grown for about 3 weeks and an inoculum containing 105 conidia per mL was sprayed with the prepared suspension until initial run-off in a humidified chamber. The disease symptoms were evaluated after one week with resistance demonstrated in all treated transgenic plants. All nonsprayed transgenic and wild type plants showed similar disease symptoms. Infected leaf samples were surface sterilized and re-cultured on V8 medium. The characteristics of the recovered pathogen confirmed that it was identical to the stock culture of W353. The same experiment has been conducted on seedlings from transgenic (T1 generations) and non-transformed plants. The non-transformed seedlings showed the first disease symptoms on their cotyledons and lower leaves. Disease resistance was observed in seedlings of the treated transformed plants as compared to nontransformed ones.
Fahrettin Goktepe and Harrison Hughes*
Fahrettin Goktepe and Harrison Hughes*
The watermelon cv. Crimson Sweet was transformed with the copper inducible isopentenyl transferase, the rate-limiting step in cytokinin biosynthesis, gene via Agrobacterium tumafaciences (LBA4404). Transformed (ipt) and nontransformed plants were regenerated from tissue culture and clonally propagated by the rooting of leaf node cuttings. Twelve plants of each were grown in 1-gal. pots. Once the plants initiated new growth both transgenic plants and wild type plants were sprayed with one of four different concentrations (0, 5, 10, & 50 μm) of CuSO4. The experimental unit was a single plant with three replicates. The growth rate, number of leaves, flowers, lateral shoots, and chlorophyll content were measured weekly for five weeks. Treated transgenic plants had greater numbers of leaves, flowers and lateral branches as well as higher chlorophyll levels. Pollen viability was examined in all treatments with no differences among treatments. Plants of both types were self pollinated to generate seeds. Female flowers were bagged before opening and then selfed. Selfed flowers were bagged for at least two days. The fruits were grown for eight to ten weeks with support. Once they reached maturity, fruits were harvested and fruit shape, flesh color, brix, number of normal seeds, number of colored but empty seeds and number of white seeds were recorded. Significant differences were observed only in seed number between wild type and transgenic (both treated and nontreated,) watermelon fruits. The number of seeds in transgenic watermelon plants treated with CuSO4 was reduced to about 5% to 7% of wild type plants. Transgenic plants which received no CuSO4 had approximately 33% to 50% of the seed of wild type.
Fahrettin Goktepe and Harrison Hughes
`Crimson Sweet' watermelon was transformed with a copper inducible ipt gene. Clonally propagated transformed and non-transformed plants were sprayed with three different concentrations (0, 50, 100 mm) of CuSO4 at the 2–3 leaf stage twice in a 24-hour period prior to their inoculation with fusarium wilt organisms. Plants were inoculated via root dip with two different isolates of Fusariumoxysporum sp. niveum Race 2. The pathogenic strains of Fusariumoxysporum sp. niveum Race 2, Fl 99-1, and Calg 15-19, were cultured on PDA solid medium and then transferred to a sterile flask filled with 50 mL of potato dextrose broth (PDB) liquid medium. These flasks were placed on a shaker at 100 rpm for 4–5 days before the inoculation date and the final concentration of 106 spores/mL was adjusted for inoculation. Roots of the experimental units were gently washed and were then infected by dipping them in to the beaker containing the isolates for 1 min. Inoculated plants were transferred to planting trays and maintained under growth chamber conditions. Plants then were watered as needed. Fusarium wilt symptoms initially appeared approximately 7–10 days after the infection. The Cu-ipt transformants exhibited a clear and significant resistance over non-transformed plants. The severity of disease development was relatively higher in the control Cu-ipt and non-transformed plants when compared to the plants treated with CuSO4. The control Cu-ipt plants were comparatively healthier than the control non-transformed plants. Infected plants were removed from soil 2 weeks after inoculation, washed and the stems were cut vertically for rating of browning of the vascular system.
Zhanao Deng, Fahrettin Goktepe, and Brent K. Harbaugh
The ornamental value of caladium (Caladium ×hortulanum Birdsey) depends to a large extent on its foliar characteristics. Efficient genetic improvement of caladium foliar characteristics requires a good understanding of the inheritance of these traits, including leaf shape, color, and spots. This study was conducted to determine the inheritance of leaf spots in caladium and to understand their relationships with leaf shape and main vein color. Eighteen controlled crosses were made among eight commercial cultivars expressing red or no leaf spots, and progeny of these crosses were observed for segregation of leaf spots as well as leaf shape and vein color. A single locus with two alleles is shown to be responsible for the presence or absence of leaf spots in caladium, with the presence allele (S) dominant over the absence allele (s). The major spotted commercial cultivar Gingerland is heterozygous for this trait. Leaf spots are inherited independently from leaf shape, but they are closely linked with the color of the main leaf veins. The recombination frequencies between the leaf spot locus and the main vein color locus ranged from 0.0% to 8.9% with the crosses or the parental cultivars used, with an average of 4.4%. Leaf spots and vein colors represent the first linkage group of ornamental traits in caladium and possibly in other ornamental aroids. The knowledge gained in this study will be valuable when it comes to determine what crosses to make for development of new cultivars. It may be also useful to those interested in determining the inheritance of similar traits in other ornamental plants, including other ornamental aroids such as dieffenbachia (Dieffenbachia Schott).
Ertan Yildirim, Huseyin Karlidag, Metin Turan, Atilla Dursun, and Fahrettin Goktepe
This study was conducted to investigate the effects of root inoculations with Bacillus cereus (N2-fixing), Brevibacillus reuszeri (P-solubilizing), and Rhizobium rubi (both N2-fixing and P-solubilizing) on plant growth, nutrient uptake, and yield of broccoli in comparison with manure (control) and mineral fertilizer application under field conditions in 2009 and 2010. Bacterial inoculations with manure compared with control significantly increased yield, plant weight, head diameter, chlorophyll content, nitrogen (N), potassium (K), calcium (Ca), sulfur (S), phosphorus (P), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) content of broccoli. The lowest yield per plant, plant weight, steam diameter, and chlorophyll content were recorded in the control, but the manure with Bacillus cereus (BC), Rhizobium rubi (RR), and Brevibacillus reuszeri (BR) inoculations increased yield 17.0%, 20.2%, and 24.3% and chlorophyll content by 14.7%, 14.0%, and 13.7% over control, respectively. Bacterial inoculations with manure significantly increased uptake of macronutrients and micronutrients by broccoli. In conclusion, seedling inoculation with BR and especially RR may partially substitute costly synthetic fertilizers in broccoli.
Zhanao Deng, Fahrettin Goktepe, Brent K. Harbaugh, and Jinguo Hu
Caladium (Caladium ×hortulanum Birdsey) is an important aroid widely used in the ornamental plant industry. Concerns have been raised about possible loss of genetic diversity due to a drastic decline in the number of cultivars in the last century. This study assessed genetic diversity and relationships among caladium cultivars and species accessions. Forty-five major cultivars and 14 species accessions were analyzed based on 297 DNA fragments produced by the target-region amplification polymorphism marker system. A low level of diversity (44.4% polymorphism) was exhibited in cultivars, while a high level of diversity (96.8% polymorphism) was present among seven accessions of Caladium bicolor (Aiton) Vent., Caladium marmoratum Mathieu, Caladium picturatum C. Koch, and Caladium schomburgkii Schott. A small percentage (7.6%) of DNA fragments was present in cultivars but absent in the seven species accessions, while a high percentage (32.2%) of DNA fragments was present in the seven species accessions but absent in cultivars. Cultivars shared a higher level of similarity at the molecular level with an average Jaccard coefficient at 0.802, formed a large group in cluster analysis, and concentrated in the scatter plot from a principal-coordinate analysis. Two accessions of C. bicolor and C. schomburgkii were very similar to cultivars with Jaccard similarity coefficients from 0.531 to 0.771, while the rest of the species accessions had small similarity coefficients with cultivars (0.060 to 0.386). Caladium steudnirifolium Engler and Caladium lindenii (André) Madison were very dissimilar to C. bicolor, C. marmoratum, C. picturatum, and C. schomburgkii, with Jaccard similarity coefficients from 0.149 to 0.237 (C. steudnirifolium) and from 0.060 to 0.118 (C. lindenii). There is a limited amount of molecular diversity in caladium cultivars, but the great repertoire of unique genes in species accessions could be used to enhance the diversity in future cultivars and reduce potential genetic vulnerability.
Zhanao Deng, Jinguo Hu, Fahrettin Goktepe, Brady A. Vick, and Brent K. Harbaugh
Cultivated caladiums are valued for their bright colorful leaves and are widely used in containers and landscapes. More than 1500 named cultivars have been introduced during the past 150 years, yet currently only about 100 cultivars are in commercial propagation in Florida. Caladium tubers produced in Florida account for 95% of the world supplies. Loss of caladium germplasm or genetic diversity has been a concern to future improvement of this plant. In addition, the relationship among the available cultivars, particularly those of close resemblance, has been lacking. This study was conducted to assess the genetic variability and relationship in commercial cultivars and species accessions. Fifty-seven major cultivars and 15 caladium species accessions were analyzed using the target region amplification polymorphism marker technique. This marker system does not involve DNA restriction or adaptor linking, but shares the same high throughput and reliability with the amplified fragment length polymorphism system (AFLP). Eight primer combinations amplified 379 scorable DNA fragments among the caladium samples. A high level of polymorphism was detected among the species accessions as well as among cultivars. These markers allowed differentiation of all the cultivars tested, including those hardly distinguishable morphologically. Clustering analysis based on these DNA fingerprints separated the cultivars into five clusters and Caladium lindenii far from other caladium species. The availability of this information will be very valuable for identifying and maintaining the core germplasm resources and will aid in selecting breeding parents for further improvement.
Fahrettin Goktepe, Zhanao Deng, Brent K. Harbaugh, Teresa Seijo, and Natalia A. Peres
Caladiums, widely used in containers and landscapes as ornamental plants for their bright colorful leaves, are generally forced or grown from tubers. Commercial production of these tubers in central Florida is through dividing “seed” tubers and growing them in fields. Tuber quality is therefore of critical importance to success in container forcing, landscape use, and tuber production. Fusarium tuber rot (Fusarium solani) has been recognized as the most-destructive disease that affects caladium tuber quality. There is anecdotal evidence from growers indicating the existence of resistance in commercial caladium cultivars. To identify and confirm the source of fusarium tuber rot resistance in caladium, F. solani isolates have been collected from rotting tubers grown under different soil conditions and from different locations. The pathogenecity of these isolates has been tested through artificial inoculation of fresh harvested and/or stored tubers, and a number of highly virulent isolates have been identified. These isolates have been used to refine inoculation and disease evaluation techniques. Two techniques, spraying a conidial suspension onto fresh cut surfaces and inserting Fusarium-infested carnation leaf segments into artificial wounds, have proven to yield consistent resistance/susceptibility ratings among cultivars of known difference in resistance to fusarium tuber rot. Appropriate incubation temperatures and humidity seem to be very critical for disease development and evaluation. The two techniques have been used to evaluate 35 cultivars. Several cultivars, including `Candidum', showed a high level of resistance to fusarium tuber rot, and may be good breeding parent for developing new resistant cultivars.
Fahrettin Goktepe, Teresa Seijo, Zhanao Deng, Brent K. Harbaugh, Natalia A. Peres, and Robert J. McGovern
Fusarium tuber rot, incited by Fusarium solani, is the major cause of losses of tuber quality and quantity in caladium (Caladium ×hortulanum) during storage and production. To develop a reliable inoculation method for evaluating cultivar susceptibility to Fusarium tuber rot and identifying sources of resistance, the effect of temperature on the mycelial growth of F. solani in vitro and on tuber rot in vivo was examined. The optimal temperature was then used to study the aggressiveness of F. solani isolates. The effect of temperature (13, 18, 23, 28, and 33 °C) on radial mycelial growth of nine F. solani isolates in vitro was determined, and all responded similarly to temperature variables, with optimal growth predicted to be at 30.5 °C. The relationship of these temperatures to disease development was then determined for the most aggressive F. solani isolate 05-20 and it was found that disease development in inoculated tubers was greatest at low temperatures (13 and 18 °C). Cold damage to tubers was observed at 13 °C; therefore, 18 °C was chosen for all future disease screening. The aggressiveness of nine isolates was tested on two caladium cultivars. Significant differences among isolates were observed for the diameter of rotted tissue in both cultivars, indicating that choice of isolate was important for screening. Isolates 05-20 and 05-257 were highly aggressive on both cultivars. Tubers of 17 commercial caladium cultivars were inoculated with three isolates (04-03, 05-20, and 05-527) and incubated at 18 °C. The interaction between isolates and cultivars was highly significant (P < 0.0001), indicating that cultivars were not equally susceptible to different pathogenic isolates of F. solani. Lesion diameters differed significantly (P < 0.0001) among cultivars/isolates and ranged from 9.5 mm (‘Rosebud’ and ‘White Christmas’ for isolate 04-03) to 23.9 mm (‘Carolyn Whorton’ for isolate 05-20). The cultivars were ranked for susceptibility to tuber rot within each isolate and the normalized total rank for the three isolates was used to place cultivars into four categories: resistant (‘Candidum’, ‘Rosebud’, ‘White Christmas’, ‘Florida Sweetheart’, and ‘Aaron’), moderately resistant (‘White Wing’ and ‘Red Flash’), susceptible (‘Candidum Jr.’, ‘White Queen’, ‘Red Frill’, ‘Florida Cardinal’, ‘Miss Muffet’, and ‘Postman Joyner’), and highly susceptible (‘Fannie Munson’, ‘Gingerland’, ‘Frieda Hemple’, and ‘Carolyn Whorton’). The availability of these sources of host plant resistance, aggressive isolates, and resistance assessment techniques will facilitate the development of new Fusarium-resistant caladium cultivars.