Progress 09/28/05 to 09/27/10
Outputs
Progress Report Objectives (from AD-416) Develop and implement a multi-disciplinary biologically based pest/disease management technologies that are environmentally acceptable and have cost/benefit ratios. Develop and implement a multi-disciplinary based technologies to ensure uniform planting seeds (slips and crowns) to maximize field establishment and to control natural flowering and reduce costs and fruit loss in a cost effective manner. Develop and implement a multi-disciplinary technology to manage fruit quality, minimizing translucency and seasonal variation in fruit quality. Approach (from AD-416) The project will utilize traditional breeding techniques and genetic engineering to develop horticulturally acceptable cultivars resistant to major disease, insects and nematodes of Hawaii's pineapple. To couple these breeding approaches to field management strategies appropriate to the new varieties multi-disciplinary biologically based pest/disease management technologies will be developed that ensure a desirable cost- benefit ratio. The project will use a basic understanding of planting material development and physiology to ensure that early field establishments and survival are maximized. The project will utilize field management and genetic engineering approaches and strategies that will enable the industry to minimize potential impact of natural flowering on yield and harvest costs. The project will develop field management strategies that utilize the latest technology to provide a year round consistent supply of high quality fruit that has a minimum of translucency and the most desirable quality. Formerly 5320-22430-021-01G (8/05). Documents SCA with U of HI Manoa. Using residual funding, the following subprojects report the following: �Natural Flowering Control via Suppression of Ethylene Synthesis�: seven independent transgenic lines of the low acid fresh fruit pineapple cultivar 73-050 and 11 independent lines of 73-114 were generated with an ACS antisense construct. Lines have survived selection on hygromycin and are GUS, PCR and Southern positive. Since funding was withdrawn in 2007, the transformed lines have not been tested for delayed flowering. �Pesticide Testing�: soil and root samples from diseased plants were collected from all areas of cultivation from pineapple plantations. Three bioassay methods were developed and compared for effectiveness in recovery of primary pathogens, Pythium spp. and Phytophthora spp. From all 3 plantations, a total of 1145 isolates of Pythium spp. were collected. From the isolates 50% were identified as Pythium splendence, 25% were identified as Pythium arrhenomonis; 9% were identified as Pythium gramanicola and the remaining 16% were unidentifiable. Of the 410 isolates of Phytophthora spp, most were found to be Phytophthora cinnamomi. Random samples of 5 isolates of each species were subjected to laboratory fungicide screening. Previcure Flex �, Previcure Energy � and Oxidate � were tested against Pythium spp. and Reason� was tested for Phytophthora cinnamomi. Only Oxidate was effective against Pythium spp. on pineapple. Reason � had no effect on controlling growth of Phytophthora cinnamomi or any of the Pythium spp. Greenhouse efficacy studies with the most promising fungicide Oxidate � will be conducted. �Non-transgenic control of virus�: a spatial and temporal study of the incidence and patterns of plants infected by pineapple mealybug with associated viruses were conducted in commercial fields to determine if particular regions of planting blocks. The incidences of PMWaV-1 and PMWaV-2 at planting, at plant crop fruit harvest at the beginning of the ratoon crop and at ratoon fruit harvest were determined for end, side and central regions of planting blocks in 8 commercial fields. At planting, incidence of PMWaV-1 and PMWaV-2 did not differ but at ratoon harvest, central regions of planting blocks had lower virus incidence at the time of ratoon fruit harvest suggesting that collection of planting propagation material from the central regions of planting blocks will help minimize the incidence of PMWaV when screening for virus free material is not feasible. Oligonucleotides specific for pineapple badnaviruses identified from pineapple in Hawaii were used to screen 2 local commercial pineapple hybrids. One hybrid that had initially been imported from 2 different sources had different mean incidences of badnaviruses (45%, 56%) depending on the source of the hybrids; the other source was only 4%. The project was monitored through telephone and email communications.
Impacts
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Develop and implement a multi-disciplinary biologically based pest/disease management technologies that are environmentally acceptable and have cost/benefit ratios. Develop and implement a multi-disciplinary based technologies to ensure uniform planting seeds (slips and crowns) to maximize field establishment and to control natural flowering and reduce costs and fruit loss in a cost effective manner. Develop and implement a multi-disciplinary technology to manage fruit quality, minimizing translucency and seasonal variation in fruit quality. Approach (from AD-416) The project will utilize traditional breeding techniques and genetic engineering to develop horticulturally acceptable cultivars resistant to major disease, insects and nematodes of Hawaii's pineapple. To couple these breeding approaches to field management strategies appropriate to the new varieties multi-disciplinary biologically based pest/disease management technologies will be developed that ensure a desirable cost- benefit ratio. The project will use a basic understanding of planting material development and physiology to ensure that early field establishments and survival are maximized. The project will utilize field management and genetic engineering approaches and strategies that will enable the industry to minimize potential impact of natural flowering on yield and harvest costs. The project will develop field management strategies that utilize the latest technology to provide a year round consistent supply of high quality fruit that has a minimum of translucency and the most desirable quality. Formerly 5320-22430-021-01G (8/05). Documents SCA with U of HI Manoa. Significant Activities that Support Special Target Populations No new funding was received during the period. Using residual funding, the following subprojects report the following: For the subproject �Natural Flowering Control via Suppression of Ethylene Synthesis�, seven independent transgenic lines of the low acid fresh fruit pineapple cultivar 73-050 and 11 independent lines of 73-114 were generated with an ACS antisense construct. Lines have survived selection on hygromycin and are GUS, PCR and Sothern positive. Since funding has been withdrawn in 2007, the transformed lines have not been tested for delayed flowering. For the subproject �Pesticide Testing,� soil and root samples from diseased plants were collected from all areas of cultivation from pineapple plantations. Three bioassay methods were developed and compared for effectiveness in recovery of primary pathogens, Pythium spp. and Phytophthora spp. From all three plantations, a total of 1145 isolates of Pythium spp. were collected. From the isolates 50% were identified as Pythium splendence, 25% were identified as Pythium arrhenomonis; 9% were identified as Pythium gramanicola and the remaining 16% were unidentifiable. Of the 410 isolates of Phytophthora spp, most were found to be Phytophthora cinnamomi. Random samples of 5 isolates of each species were subjected to laboratory fungicide screening. Previcure Flex �, Previcure Energy � and Oxidate � were tested against Pythium spp. and Reason� was tested for Phytophthora cinnamomi. Only Oxidate was effective against Pythium spp. on pineapple. Reason � had no effect on controlling growth of Phytophthora cinnamomi or any of the Pythium spp. Greenhouse efficacy studies with the most promising fungicide Oxidate � will be conducted. For the subproject, �Non-transgenic control of virus control,� a spatial and temporal study of the incidence and patterns of plants infected by pineapple mealybug will associated viruses were conducted in commercial fields to determine particular regions of planting blocks were more desirable areas for harvesting propagation materials. The incidences of PMWaV-1 and PMWaV-2 at planting, at plant crop fruit harvest at the beginning of the ratoon crop and at ratoon fruit harvest were determined for end, side and central regions of planting blocs in eight commercial fields. At planting incidence of PMWaV-1 and PMWaV-2 did not differ but at ratoon harvest, central regions of planning blocks had lower virus incidence at the time of ratoon fruit harvest suggesting that collection of planting propagation material from the central regions of planting blocks will help minimize the incidence of PMWaV when screening for virus free material is not feasible. Oligonucleotides specific for pineapple badnaviruses identified from pineapple in Hawaii were used to screen two local commercial pineapple hybrids. One hybrid that had initially been imported from two different sources had different mean incidences of badnaviruses (45%, 56%) depending on the source of the hybrids. The incidence of badnavirus in the other source was only 4%. ADODR monitored this project through email and telephone communications and progress report.
Impacts
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Develop and implement a multi-disciplinary biologically based pest/disease management technologies that are environmentally acceptable and have cost/benefit ratios. Develop and implement a multi-disciplinary based technologies to ensure uniform planting seeds (slips and crowns) to maximize field establishment and to control natural flowering and reduce costs and fruit loss in a cost effective manner. Develop and implement a multi-disciplinary technology to manage fruit quality, minimizing translucency and seasonal variation in fruit quality. Approach (from AD-416) The project will utilize traditional breeding techniques and genetic engineering to develop horticulturally acceptable cultivars resistant to major disease, insects and nematodes of Hawaii's pineapple. To couple these breeding approaches to field management strategies appropriate to the new varieties multi-disciplinary biologically based pest/disease management technologies will be developed that ensure a desirable cost- benefit ratio. The project will use a basic understanding of planting material development and physiology to ensure that early field establishments and survival are maximized. The project will utilize field management and genetic engineering approaches and strategies that will enable the industry to minimize potential impact of natural flowering on yield and harvest costs. The project will develop field management strategies that utilize the latest technology to provide a year round consistent supply of high quality fruit that has a minimum of translucency and the most desirable quality. Formerly 5320-22430-021-01G (8/05). Documents SCA with U of HI Manoa. Significant Activities that Support Special Target Populations Detecting disease organisms from soil and roots through bioassay: Root rots caused by Phytophthora and Pythium cause severe losses. There is no current assay to detect these organisms in soil or roots. Soil and root samples from diseased plants were collected from all areas of cultivation from Maui Pineapple Plantation, joining samples collected the previous year from Dole and former Del Monte plantation. A total of 420 samples have been collected and are in storage. Australian research has indicated that there is evolving a strain of Phytophthora cinnamomi, one of the causative agents for root rot, that has become resistant to Alliete �, the major fungicide in use by the pineapple industry. A bioassay to identify Alliete �-resistant P. cinnamoni has been developed. Screening for Phytophthora cinnamomi and Phytophthora nicotiana found in diseased pineapple samples is underway. Pythium isolates are also being screened. Insect Integrated Pest Management on Pineapple: Significant pineapple damage is caused by the Tineid moth, Opogona sacchari. Primary damage to the pineapple plant is to cause gummosis to the fruit, which causes fruit discoloration; if severe, rejection of the fruit. Sampling plans were developed to identify degree of infestation in pineapple fields. Three predatory wasps, Trichogramma achaeae, T. papilionis, and T. chilonis shows strong potential as biological control agents, to kill the moth eggs before hatching. Potential pheromone attractants are being investigated cooperatively with ARS. Non-transgenic methods for Pineapple Mealybug Wilt Associated Viruses (PMWaVs): Ants tend grey pineapple mealybugs, the vector for Pineapple mealybug wilt associate ampeloviruses. Previous studies have shown that broadcast or bait stations with Amdro Pro � can reduce the spread of PMWaVs. In ratoon-aged pineapple fields, bait stations to control ants within the crop did not reduce virus spread. Bait stations along field borders prevented ants from moving into the ratoon crop, but if ant colonies were already established in the field, bait stations provided no control over ants, mealybugs and did not reduce the spread of PMWaVs. A real time PCR assay has been developed to monitor virus concentrations in mealybugs and pineapple plants. PMWaV-3, a virus previously identified at low incidence in the traditionally grown hybrids and cultivars in Hawaii, was present in about 70% of one of the new hybrids. Incidences of PMWaV-2 were nearly 85% in the accessions in Hilo, HI. Such high incidences indicate the need for screening of plant material prior to mass propagation and further planting expansions. Preliminary results show that the grey pineapple mealybugs remain infective with PMWaVs for only 24 hours after virus acquisition. Trap crops may provide an opportunity to reduce the spread of PMWaVs. The project is monitored through email, telephone communications and meetings.
Impacts
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Publications
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Develop and implement a multi-disciplinary biologically based pest/disease management technologies that are environmentally acceptable and have cost/benefit ratios. Develop and implement a multi-disciplinary based technologies to ensure uniform planting seeds (slips and crowns) to maximize field establishment and to control natural flowering and reduce costs and fruit loss in a cost effective manner. Develop and implement a multi-disciplinary technology to manage fruit quality, minimizing translucency and seasonal variation in fruit quality. Approach (from AD-416) The project will utilize traditional breeding techniques and genetic engineering to develop horticulturally acceptable cultivars resistant to major disease, insects and nematodes of Hawaii's pineapple. To couple these breeding approaches to field management strategies appropriate to the new varieties multi-disciplinary biologically based pest/disease management technologies will be developed that ensure a desirable cost- benefit ratio. The project will use a basic understanding of planting material development and physiology to ensure that early field establishments and survival are maximized. The project will utilize field management and genetic engineering approaches and strategies that will enable the industry to minimize potential impact of natural flowering on yield and harvest costs. The project will develop field management strategies that utilize the latest technology to provide a year round consistent supply of high quality fruit that has a minimum of translucency and the most desirable quality. Formerly 5320-22430-021-01G (8/05). Documents SCA with U of HI Manoa. Significant Activities that Support Special Target Populations This report documents research conducted under an SCA between ARS and the UH Manoa. Additional details can be found in the report for parent project 5320-22430-021-00D, Detection, Control and Area wide Management of Fruit Flies. Nematode Control The reproduction of and damage caused by the root-knot nematode Meloidogyne javanica was compared on pineapple, Ananas comosus, transformed and expressing the Oc-I'D86 cystatin to wild type pineapple. The wild type pineapples were heavier than the transgenic plants at planting and at harvest. Plant growth was unaffected by nematodes. The nematode populations were greater on the transgenic plants than on the wild type plants (2295 vs 1596 eggs). The number of eggs/g dry root was higher on the transgenic plants than on the wild type plants (1574 vs 965 eggs/g dry root). The number of eggs/plant was greater in the wild type plants for the first 3 weeks of harvest, but for the remaining 4 weeks the number of eggs/plant was greater in the transgenic line. Virus Control Development of transgenic pineapple plants with virus resistant genes Transformation with Agrobacterium tumefaciens was used to introduce gene constructs within a pCAMBIA backbone that contain either 1) the coat protein gene (CP) of Pineapple mealybug wilt associated virus-2 (PMWaV-2), or 2) a sequence composed of a portion of the CP gene in sense orientation, a portion of the HSP70h gene in sense orientation, and a portion of the CP gene in sense and antisense orientations aligned in tandem to form a hairpin-like structure of complementary sequences were tested. PCR analyses using oligonucleotide pairs specific for the CP region or CP antisense sequence and the NOS-terminator region of 105 putatively transformed plants in the greenhouse showed that 8 plants, representing up to 7 distinct lines are positive for the transgene. Development of a direct somatic embryogenesis system for pineapple regeneration Stems, inflorescences, and leaf-bases of pineapple plants were evaluated for use in thin cell-layer direct embryogenesis. Direct embryogenesis was successfully achieved using stem-bases. However, the results obtained using this system varied depending upon 1) the tissue turgidity, which was highly influenced by the hybrid and media used, 2) the difficulty in obtaining uniform thin cell layers, and 3) the variability in hormone levels along the length of the stems. Embryos that were produced and germinated using this system have developed into plants that are currently being evaluated for agronomically undesirable traits under greenhouse conditions. To date, all appear normal. The efficiency of direct embryogenesis using thin cell-layers derived from leaf bases was 30-50%, when TDZ was included in the media. Callusing of the explants and regeneration into plantlets were limiting factors. This was circumvented by utilizing a media that promoted adventitious bud induction which we had developed during our direct embryogenesis studies, and by modifying the system to capitalize on this highly efficient adventitious bud generation system. The project is monitored through meetings, email, telephone communications, and on-site visits.
Impacts
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report documents research conducted under a Specific Cooperative Agreement between ARS and the University of Hawaii at Manoa. Additional details of research can be found in the report for the associated in- house CRIS 5320-22430-021-00D, Detection, Control and Area wide Management of Fruit Flies. Agrobacterium-mediated transformation of pineapple for nematode resistance and flowering control. Two transformation systems were developed for transformation of pineapple for nematode resistance and flowering control. In both cases, the low acid pineapple variety MD-1 was used as source plants. The construct for nematode resistance was a modified rice cystatin (ubi9-d86), 35S-GUS, 35S-Hpt (hygromycin resistance). The Agrobacterium was AGL0 and stem segments and leaf bases were the plant tissues infected; regeneration was via an adventitious bud pathway. For nematode resistance, a total of 22 PCR positive transgenic lines were
produced. Over 300 individuals of line 101 are in greenhouse for nematode bioassays. All other lines have gone through at least 4 rounds of selection in hygromycin. All lines except for two have been transferred to rooting media containing hygromycin. For flowering control, the construct for flowering control was ACC antisense (ubi9), 35S-Hpt (hygromycin resistance). The promoters are constitutive. Agrobacterium AGL0 and stem segments and leaf bases were the plant tissues infected; regeneration was via an adventitious bud pathway. A total of 7 PCR positive transgenic lines were produced. Two additional lines have completed 3 rounds of testing and require further testing. Line 51 (over 300 individual plants) is being tested in greenhouse for bioassay of flowering control. Other lines have gone through at least 4 rounds of selection in hygromycin and are being multiplied in media with hygromycin. Using adventitious bud pathway, an Agrobacterium-mediated transformation method has
been developed for pineapple. The method is highly repeatable and the transformation efficiency is about 1 transgenic line per thousand pieces of tissue used.
Impacts
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