Control of Bacterial Wilt Disease of Ginger through an Integrated Pest Management Program
Edible ginger (Zingiber officinale) is a major export crop on Hawaii Island. Bacterial wilt caused by Ralstonia solanacearum is the crop’s most problematic disease. During Year 2 of this project, we have:
a) grown and distributed 400 pounds of wilt-free ginger seed pieces to commercial growers, educators and backyard growers;
b) shown that hydroponically-grown ginger should be grown without additional shade under rain shelters;
c) conducted a study on use of hot water treatment to kill R. solanacearum in naturally-infected rhizomes;
d) conducted two pot studies on the use of alternative methods to control ginger wilt (e.g. vermicompost, vermicompost tea and indigenous microorganisms);
e) demonstrated the usefulness of the Enrichment-PCR method to detect R. solanacearum in field soil or media and convinced the University of Hawaii’s Agricultural Diagnostic Service Center to adopt this protocol;
f) demonstrated that planting wilt-free ginger seed pieces into a field that tested free of R. solanacearum can result in good yields, although there still may be pockets of the pathogen;
g) conducted an informational meeting for growers;
h) completed two segments of an educational video;
i) presented information on the health benefits of ginger to the public; and
j) made five scientific presentations at two scientific meetings held in Honolulu, Hawaii and Waikoloa, Hawaii.
The overall goal of this project is to develop and demonstrate sustainable farming practices that control bacterial wilt in edible ginger.
Specific objectives are to:
1) demonstrate the importance of clean planting materials;
2) demonstrate procedures to test fields for Ralstonia solanacearum;
3) conduct field studies to determine the effectiveness of green manure crops or rotational crops for pathogen control;
4) conduct greenhouse studies to determine the effectiveness of vermicomposts to control R. solanacearum;
5) conduct economic analysis of sustainable, farming practices; and
6) disseminate information and enhance farmer adoption of practices through a video and a web site.
From May 2011 through November 2011 to February 2012, ginger was hydroponically grown (Figure 1), then harvested, washed and packed in 16 boxes (25 pounds each) for distribution as wilt-free seed pieces. These ginger plants were free of Ralstonia solanacearum, because earlier they had been cleaned of the pathogen during tissue-culture, and then multiplied in disease-free medium using hydroponic culture.
Informational meeting for growers.
On March 6, 2012, we held an evening meeting for commercial ginger farmers, back yard ginger growers and students. There were 28 growers in attendance. Presentations were made by: a) Dr. Shintaku on the wilt disease caused by Ralstonia solanacearum; b) Dr. Arancon on results of a pot study that used vermicompost to control bacterial wilt of ginger; c) graduate student Ms. Sharon Motomura on Enrichment-PCR testing for bacterial wilt of ginger in soil and medium; d) Junior Researcher Ms. Ferol White on results of a field trial in which clean ginger seed pieces were planted in a field that had tested free of ginger wilt; and e) Dr. Bernie Kratky on effects of shade on hydroponically-grown ginger. The presentations were also video-taped and DVDs distributed upon request to those unable to attend.
Side-by side photographs of two ginger seed pieces (one with wilt and the other without) were shown to growers, and they were asked to guess which one was disease-free (Figure 2). It was clearly demonstrated that it is difficult to distinguish visually between wilt-free ginger seed pieces and those infected by wilt during the early stages. The only way that growers can be assured of disease-free ginger seed pieces is by multiplying rhizomes that were originally tissue-cultured using soil-less medium or hydroponic culture.
During this meeting, we distributed wilt-free ginger seed pieces (15 x 25 pound boxes) to commercial or educational cooperators on the Islands of Hawaii and Maui. In addition, one box each was sent to a cooperating researcher at Texas A & M University, and another to a cooperating commercial nursery operator in Florida. Finally, we distributed 40 bags (4-6 ounces) of clean ginger seed pieces to backyard growers, along with instructions on “How to Multiply Disease-Free Ginger in pots.”
Dr. Kratky, Dr. Miyasaka and Ms. White presented a poster entitled “Production of edible ginger clean seed by sub-irrigation methods in Hawaii” at the American Society of Horticultural Science during September 25-28, 2011 at Waikoloa, Hawaii (Figure 3).
Effect of shading on hydroponic production of ginger.
Dr. Kratky conducted a hydroponic study to determine the effect of shading on production of ginger rhizomes. Treatments consisted of four shading levels by placing 0, 30, 47 and 80% shade screen material under the plastic film cover of the rain shelters. He found that the highest yields occurred without shading (Table 1). Dr. Kratky is in the process of writing this report for publication by the University of Hawaii’s College of Tropical Agriculture and Human Resources.
Effect of silicon on hydroponic production of ginger.
Currently in the greenhouse, we are conducting a trial to determine whether the addition of silicon will improve hydroponic production of ginger.
Continued selection of tissue-cultured ginger.
In addition, we are continuing the process of tissue-culturing disease-free ginger plants, by growing them in pots (Figure 4), followed by selection for size and uniformity of rhizomes during hydroponic culture.
Hot water seed piece treatment.
Edible ginger is vegetatively propagated, and therefore, the incidence of replanting pathogen-infested rhizomes is likely when farmers lack the ability to obtain non-infested planting material. A hot water treatment trial of infected seed pieces began in February 2012 by cooperator Dr. Marcel Tsang (University of Hawaii – Hilo) to determine a treatment regime that could kill R. solanacearum within seed pieces with minimal reduction in germination.
Seed pieces that were R. solanacearum-infested (yet otherwise healthy) were selected for use in this trial using Polymerase Chain Reaction (PCR) to detect the presence of the pathogen. Seed pieces were completely submerged in a hot water bath until their internal temperature reached 50?C (which took approximately 20 minutes), and then additional treatments included additional 4-5 minute increments up to a total of 55 minutes (Table 2).
Our results showed a decrease in viable R. solanacearum populations as a direct result of prolonged treatment time except for the final and longest treatment of 55 minutes (Table 2). This anomalous result is being investigated further whether it was caused by contamination by other races of R. solanacearum. We plan on planting out ginger seed pieces from the last two treatments in pots to monitor their growth and possible development of disease symptoms. In addition, rhizomes will be re-tested using strain-specific primers in PCR analysis.
Figure 5 shows wilt-infected ginger plants in the field. We have shown that our methodology of bacterial extraction from soil followed by Enrichment-PCR is sensitive, reliable and cost-effective. This methodology has been adopted by the Agricultural Diagnostic Service Center at the University of Hawaii at Manoa, in Hilo, Hawaii. The center now offers testing of R. solanacearum in soil and other media, in addition to Enzyme-Linked Immunoassay (ELISA) testing of tissue samples.
Testing of these soil samples using enrichment-PCR not only yields a positive or negative result but can provide growers the added information of whether or not the bacteria found is dead or alive. In addition, it allows us to determine relative concentrations of bacteria present within a sample according to the time at which the amplified DNA is visible as a band during the five day period of enrichment. At the highest concentration of 106 (Colony Forming Unit) CFU, the band appears at 24 hrs; at 104 CFU, it appears at 48 hrs; and at 100 CFU, the band appears at 72 hours. However, the limitation of this method is that it uses a very small volume of soil. Ralstonia solanacearum often occurs in small pockets in the field and could be missed during sampling.
At the American Phytopathological Society, Ms. Motomura, Dr. Arancon, Dr. Shintaku and Dr. Miyasaka presented a poster entitled “Detection of Ralstonia solanacearum in Hawaiian field soils and evaluation of composts for suppressing pathogen populations” during August 6-10, 2011. At the American Society of Horticultural Science during September 25-28, 2011 at Waikoloa, Hawaii, Ms. Motomura, Dr. Arancon, Dr. Shintaku and Dr. Miyasaka presented a poster entitled “Effect of Composts on Field Soils Affected by Bacterial Wilt of Edible Ginger in Hawaii.”
To demonstrate the importance of planting wilt-free ginger seed pieces into wilt-free fields, two commercial growers agreed to cooperate in on-farm field trials. Prior to planting, Dr. Shintaku and his graduate student Ms. Motomura tested both fields using their Enrichment-PCR method and found them to be wilt-free. Wilt-free seed pieces of ginger were planted in two separate farms on the Island of Hawaii (Paukaa and Pepeekeo) during March to April 2011, and harvested during January 2012.
In the Pepeekeo on-farm trial, six rows (20 feet long x 5 foot wide) were planted side-by-side (Figure 6). Although the soil had been found to be wilt-free using the Enrichment-PCR method, one plant in the field was found to have ginger wilt during August 2011; it was removed from the field. At harvest, the row with the removed diseased plant was found to have infected rhizomes based on PCR although they appeared symptom-less. Average marketable yields (fresh weight) of three disease-free rows (excluding two border rows) were 54,030 pounds per acre (60,680 kg/ha) with 20% loss due to unmarketable yields (Table 3; Figure 7). In the row with ginger wilt (row 5), the marketable yield was only 16,600 pounds per acre (18,640 kg/ha) with 69% loss due to unmarketable yields.
No harvest data was taken from the second on-farm field trial, due to miscommunication between the cooperator and his field workers. The plots were harvested prior to any measurements of yield.
A second year field trial was planted on-farm at the Pepeekeo site on April 16, 2012. Again, the field site tested free of ginger wilt, and hydroponically grown, clean ginger seed pieces were planted. The second cooperator at Paukaa decided not to grow ginger during 2012.
In addition, another on-farm field trial has been planted at Pepeekeo, Hawaii on April 16, 2012. The objective of this second trial is to reduce air and soil temperatures to simulate growing conditions at higher elevations where ginger wilt is not a serious threat. It is hoped that the change in environmental conditions will reduce or prevent the development of the disease. Aluminet shade (40% shade) has been placed over the row where R. solanacearum had been detected in the previous on-farm study.
Effect of vermicompost on ginger and ginger wilt disease.
Dr. Arancon and graduate student Paul Flessner conducted this pot study starting May 23, 2011 and harvested on October 30, 2011. Ginger propagules (approximately 2-4 ounces or 50 – 100 g) were sown directly into one gallon pots containing media amended at different rates with vermicomposts produced by Perionyx excavatus from food wastes and shredded paper [0 (control), 20, 40, 60, 80, 100 percent substitution rates]. Potting media (Promix BX) and vermicompost were screened for Ralstonia, showing no previous contamination. Half the pots were inoculated with 108 CFU of R. solanacearum race 4 in 5 ml of water. All 12 treatments (six vermicompost rates x two pathogen rates) were replicated five times and laid out in a randomized complete block design. Plant height and disease symptoms were recorded weekly throughout the growing period. Rhizome yield and quality were recorded at harvest. Presence of R. solanacearum was assessed using the Enrichment-PCR method using race 4 specific primers. Rhizome yield was analyzed with ANOVA using Minitab 16.1.
The results of the PCR analysis showed that despite inoculation with R. solanacearum, all samples tested negative for Ralstonia. Yet, symptoms (e.g., yellowing of leaf tips and margins) were present and highest among 80% and 100% treatments in both inoculated and non-inoculated groups (Figure 8). These symptoms could possibly be due to phytotoxicity caused by high electrical conductivity (EC) as a result of high application rate of vermicomposts.
Ginger growth rate and rhizome yield both varied between treatments; highest yields and growth rate were found between 20% and 80%, while lowest yields and growth were found in the control and 100% treatment (Figure 9). Total rhizome weight was highest in the 20% vermicompost treatment. The 100% vermicompost treatment resulted consistently in the worst performing plants in this trial; they displayed the slowest and smallest growth, lowest rhizome yields and phytotoxicity symptoms (yellowing of leaf margins and tips; Figure 8). The 20% vermicompost treatment resulted in the fastest and healthiest growth and the highest rhizome yield.
The negative PCR results raised many questions as to the mechanism of suppression in all treatments. Perhaps, the media used (Promix BX) was itself suppressive. Also the high temperatures in the greenhouse coupled with the use of conventional black pots could have exceeded the acceptable range for survival of Ralstonia. While these circumstances were problematic for our research, they warrant further investigation and could potentially lead to an alternative disease suppression strategy.
A second trial is planned for June 2012. Field soil will be used in the study of the effects of vermicompost, along with improved irrigation and efforts to maintain temperatures within the ideal range for the bacterial wilt pathogen. Also, sampling of the media and analyzing with R. solanacearum test strips throughout the trial will be used to monitor the presence of the bacterium. Using information from the first trial, it will also be possible to test several alternative treatments: peat based media, high temperatures and UV exposure.
Effects of vermicompost, vermicompost tea and indigenous microorganisms on the pathogen.
A pot study was performed by Ms. Motomura under the supervision of Dr. Shintaku to determine whether vermicompost, vermicompost tea or a preparation of indigenous microorganisms (IMO) could help to reduce populations of the ginger wilt pathogen R. solanacearum in naturally infested field soil. A twelve week trial began on June 16, 2011 and ended on August 25, 2011.
Soil samples were taken and relative populations of R. solanacearum were determined using the enrichment-PCR method. A randomized block design with four replicates of each treatment (Table 4) was used. Weekly soil samples were collected from 2L pots and enriched over a five day period with aliquots collected at 0, 24, 48, 72 and 96 hours, and then analyzed using PCR.
None of the treatments showed a decrease in viable R. solanacearum populations throughout the 12 weeks. Rather, vermicompost tea at the mid and high rate showed a marked increase at weeks 9 and 12, while other treatments showed fluctuations in viable R. solanacearum populations from week to week, with no apparent trend (Figure 10).
In conclusion, vermicompost, vermicompost tea or Indigenous Microorganisms (Imolizer) did not show an antagonistic effect on bacterial populations in soil in pots. The use of enrichment–PCR in determining the presence of R. solanacearum in soil is straight-forward, although bacterial populations can only be estimated as relative levels. Analysis of these samples will be conducted later using real-time PCR to better resolve DNA levels of the pathogen in our samples.
Wilt-free ginger seed pieces were distributed to commercial growers and backyard growers during 2011. Ms. Ferol White contacted the growers and conducted a survey to determine their use of these clean ginger seed pieces if they had received ones from 2011. Of the nine growers who said that they had received previously a box of wilt-free ginger, two had grown these rhizomes in “virgin” land that had never been planted before in ginger; both farmers stated that their ginger was growing well. Two farmers grew their wilt-free ginger seed pieces only in pots; one found that an elevation of 2,800 feet was too cold for good growth of ginger, and the other stated that their plants grew well at an elevation of 650 feet. Four other farmers stated that they had grown their wilt-free ginger partly in pots and partly in the ground or soil-based medium. Of these, one farmer thought that the ginger planted in the ground appeared to have ginger wilt disease. His comment was that there was “no more good land to plant ginger…”
The video will consist of five segments: 1) hydroponic production of clean ginger seed pieces; 2) soil testing using enrichment-PCR; 3) field planting, cultivation and harvest; 4) ginger wilt disease including symptoms; and 5) specific recommendations based on scientific data. The first two segments have been videotaped, written, recorded and edited. The third has been videotaped and is being scripted for narration.
A link to the video segment on production of clean ginger seed pieces is at: http://www.youtube.com/watch?v=e8On38-LMfA. A link to the video segment on the enrichment-PCR method is at: http://www.youtube.com/watch?v=EzxVSCtXdJM. The last three segments are still in progress.
Impacts and Contributions/Outcomes
We demonstrated that by planting wilt-free ginger seed pieces into a field that tested free of ginger wilt, we were able to harvest an average of 54,030 pounds per acre (60,680 kg/ha) of marketable yield with 20% loss due to unmarketable yields. Unfortunately, the Enrichment-PCR method did not detect R. solanacearum in one location in the field, probably due to its occurrence in a small pocket. This presence of disease reduced marketable rhizome yields in one particular row by 70%. These infected rhizomes did not appear to be diseased (Figure 2); if planted by farmers during the next cropping cycle, then the new field will become infected by ginger wilt. An informational meeting for commercial growers and backyard gardeners demonstrated that these infected rhizomes could not be visually distinguished from non-infected rhizomes. The only methods to distinguish between the two are either an enzyme-linked immunoassay (ELISA) test or a PCR analysis. One way to ensure planting of wilt-free ginger seed pieces is to produce rhizomes in a wilt-free medium, either hydroponically or in pots.
Farmers and backyard growers will now be able to test their soil or medium for the presence of ginger wilt, using the Enrichment-PCR method that we developed. The University of Hawaii’s Agricultural Diagnostic Service Center has adopted this protocol for testing of soil.
During Year 2, we distributed 13 boxes of wilt-free ginger seed pieces to commercial growers, educators and backyard gardeners in Hawaii. In addition, we shipped one box of clean ginger seed pieces to Dr. Mengmeng Gu, a researcher at Texas A & M and a second box to a commercial nursery in Florida. There is increased interest in growing ginger outside of the state of Hawaii.
Promoting health benefits of ginger.
Mealani’s A Taste of the Hawaiian Range and Agriculture Festival was held on September 30, 2011. Ms. White produced an educational poster on the health benefits of ginger and a display of many ginger products (Figure 11). Cooperator William Tocantins donated bottles of Ginger Elixir for the public to sample (Figure 12).
Kratky, B.A., S.C. Miyasaka, and F. White. 2011. Production of edible ginger clean seed by sub-irrigation methods in Hawaii. Presentation (052) at American Society of Horticultural Science (ASHS), Sept. 25-28 2011, Waikoloa, HI. Available at: http://ashs.org/downloads/2011ASHS_Conference_abstracts.pdf . Accessed 11 June 2012.
Motomura, S., A. Read, N. Arancon, S. Miyasaka, and M. Shintaku. 2011. Detection of Ralstonia solanacearum in Hawaiian field soils and evaluation of composts for suppressing pathogen populations. Poster session presented at the American Phytopathological Society (APS), 6-10 Aug. 2011, Honolulu, HI.
Motomura, S., Read, A., Arancon, N., Miyasaka, S., & Shintaku, M. (2011) Effect of Composts on Field Soils Affected by Bacterial Wilt of Edible Ginger in Hawaii. Presentation (276) at American Society of Horticultural Science (ASHS), 25-28 Sept. 2011, Waikoloa, HI. Available at: http://ashs.org/downloads/2011ASHS_Conference_abstracts.pdf . Accessed 11 June 2012.
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