Management of Banana Bunchy Top in Hawaii

Final Report for SW04-064

Project Type: Research and Education
Funds awarded in 2004: $90,458.00
Projected End Date: 12/31/2008
Region: Western
State: Hawaii
Principal Investigator:
Dr. Cerruti R. R. Hooks
University of Maryland
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Project Information

Abstract:

Hawaii banana plantings are plagued with several yield reducing pests. Among which, Banana bunchy top virus (BBTV) and its aphid vectors (Pentalonia nigronervosa) are of greatest economic concern. In the pass, limited field research and outreach efforts were conducted in Hawaii with regards to these organisms. As a result, several “grower myths” developed in Hawaii regarding their management and changes in production practices resulting from these gratuitous beliefs helped to promote virus spread. Thus, the goal of this project was to help banana growers sustain the economic viability of their operations through the obtainment and dissemination of practical and trustworthy information on BBTV and Pentalonia nigronervosa that could readily be used as part of an integrated disease management program.

Project Objectives:
  1. obtaining a better understanding of the banana aphid’s biology and ecology,studying the pattern of banana bunchy top virus spread in commercial fields, and

    determining disease parameters that are important to the development of virus management practices

Introduction:

Hawaii ranks number one within the United States in banana production (Musa sp.). Annual production in Hawaii for the year 2004 was 16.5 million pounds worth $8.1 million (National Agricultural Statistics Service 2005) putting it among the states top tropical fruits. Commercial banana production occurs on all major islands with 80% of the production concentrated on the islands of Hawaii and Oahu (Constantinides and McHugh, 2003).

Banana bunchy top disease (BBTD), caused by Banana bunchy top virus (BBTV), is one of the most economically important diseases of bananas in most producing regions (Dale & Harding 1998). Plants infected early with BBTV do not bare fruit and fruits of later infected plants are typically unmarketable. Additionally, the virus spreads to suckers via the rhizome and thus the entire banana mat eventually becomes infected (Dale & Harding 1998). BBTV is transmitted by an aphid vector, Pentalonia nigronervosa (Magee 1927, Hu et al., 1996). The virus was first reported in Hawaii in 1989 (Ferreira 1991) and has since progressively spread throughout banana growing areas along the island chain. In Hawaii, banana showed a 27 and 26% reduction in yield and harvested acreage, respectively, from 2003 to 2004 (National Agricultural Statistics Service 2005). Much of this continual decline is attributed BBTV.

Since BBTV was first documented in Hawaii, concerns regarding its negative economic impact have mounted. In March 2003, a workshop was conducted in Honolulu, HI to establish research priorities for banana production in Hawaii. Concerns regarding the economic sustainability of Hawaii’s banana industry as a result of BBTV dominated the workshop (www.ipmcenters.org/pmsp/idex.cfm). Recently there has been a greater commitment to studying BBTV in the field, but the information obtained from these studies has not been well disseminated. Thus, growers and other stakeholders have continued to rely on their own guesswork regarding how to manage BBTV. As a result, several “growers’ myths” exist in Hawaii regarding BBTV and its aphid vector. Changes in production practices and the use of home remedies resulting from some of these gratuitous beliefs are fueling disease spread. Thus, leading constraints to greater adoption of reputable IPM practices in banana fields are high acceptance of untested beliefs by banana stakeholders and failure of researchers to disseminate creditable information.

In Hawaii, the amount of acreage devoted to banana has significantly decreased over time (National Agricultural Statistics Service 2005.) This reduction has been contributed partially to a lack of knowledge regarding management of BBTV and its aphid vectors. Thus “farmers’ myths” regarding BBTV has not been practical in lessening its economic severity but instead has resulted in some contemptible production practices that have contributed to its continuous spread. In some banana farming communities, the percentage of plants infected with BBTV have reached such high levels that growers are destroying their banana orchards and replacing them with secondary crops that are not as lucrative as bananas.

Cooperators

Click linked name(s) to expand
  • Rodrigo Almeida
  • Khamphout Chandara
  • Scott Chun
  • Richard Ha
  • Larry Jefts
  • Eden Perez
  • Alec Sou
  • koon-Hui Wang
  • Mark Wright

Research

Materials and methods:

Within-Plant Distribution and Binomial Sampling of Pentalonia nigronervosa (Hemiptera: Aphididae) on Banana

Sampling Locations: We sampled for P. nigronervosa on commercial banana plantations on the island of Oahu, HI. We chose sampling locations that allowed for representation of the different climatic conditions of the island, most notably the typical difference between the wet, eastern side and the dry, western side of the island. For example, the
average daily temperature at Waianae is 22.8_C, and the average annual precipitation is 77.2 cm versus an average daily temperature of 20.6_C and an average annual precipitation of 145.5 cm at Kahuku (Fig. 1, location 5) (National Weather Service 2006). These climatic regions also match banana growing areas on other Hawaiian islands. Young and Wright (2005) showed that populations of P. nigronervosa do not fluctuate significantly throughout the year; thus, we sampled over a period of 7 mo (March
September 2005). Only plants of the Dwarf Brazilian variety (Musa AAB) were sampled. Cultural practices were similar on all plantations; banana mat spacing of 2 m between plants and between rows. Every other row was wider (~ 4 m) to allow farm vehicles to pass. Plants were maintained on a drip irrigation system. Most growers did not use any chemical control for P. nigronervosa. Only one of the plantations sampled, Ewa west, had been sprayed for insect pests before sampling (~ 3 mo before our 25 March 2005 sampling date).

P. nigronervosa is found in greater numbers on suckers rather than on mature banana plants (Young and Wright 2005), although we also have observed aphids on plants bearing fruit (J.D.R., unpublished data). In terms of development of a practical sampling procedure, suckers are more useful than mature plants for reasons of accessibility for samplers. Thus, our decision to sample only smaller plants was made based on the distribution of aphids on banana and the goal of developing a user-friendly plan for growers. We quantified within-plant distribution of aphids by using a diagram representative of a small banana plant. On this diagram, leaf 1 represents the newest, unfurled, uppermost leaf (commonly referred to as the “cigar leaf”); leaves 2 to 5 are the next fully expanded leaves; leaf 6 represents all thin, blade-like leaves below the fully formed leaves; and leaf 7 represents the dry bract around the base of the pseudostem near soil level. The tip of dry bracts is composed primarily of dry tissue; however, the base of the leaf connected to the pseudostem remains hydrated. In addition, aphids are often found under this bract feeding on the pseudostem rather than on the bract itself. Banana morphology and terminology followed Robinson (1996). Because plants varied in size and leaf number, we sampled plants with variable numbers of leaves. All plants had a top and bottom leaf (positions 1 and 7) but the number of leaves in between varied. We randomly chose rows within a field and treated them as transects through the plantation. The number of rows sampled ranged from 10 to 30 depending on the size of the plantation. Along a row, all plants that were 1.5 m and less in height were sampled. When sampling a plant, the cigar leaf was first examined, then we moved down the plant, examining the spaces between the petioles and the pseudostem where aphids are located (van Hoof 1962). The cigar leaf was designated as leaf 1; the next oldest leaf down was designated leaf 2, and so on. If the plant was very small (i.e., ~ 30 cm in height), we omitted leaf positions between leaves 1 and 7. For example, many of the smallest plantlets had only leaves 1, 6, and 7; unfurled leaves were absent. Leaves were usually pulled gently away from the pseudostem to determine the presence of P. nigronervosa. We peeled leaves completely away from the pseudostem in some instances when the grower involved gave permission for this more destructive sampling method. Aphids could be found equally as well using either technique, but we found the latter to be less time consuming. Proportion of leaf-sheaths harboring aphids at different positions was compared using a 2 x 7 contingency table analysis with leaf position as the categories and frequency of encounter of aphids or absence as the contingencies.

Because we were sampling for an efficient aphid vector (Hu et al. 1996) and not a direct pest, we used a binomial sampling approach, with aphids being designated as present or absent from a plant. We used data obtained on surveys for within-plant distribution of the aphid to test our sampling plan. Enumerative field sampling for P. nigronervosa has been done (Young and Wright 2005), but it is likely to be difficult and time-consuming for producers because of the pest small size, potential for large infestations, and cryptic nature. Sequential sampling is one of the most efficient sampling schemes for making control decisions in an integrated pest management program (Romoser and Stoffolano 1998).

Effect of Banana bunchy top virus Infection on Morphology and Growth Characteristics of Banana

Three field trials were conducted to assess the impact of BBTV on banana growth and determine BBTD’s incubation period. In 2005, field experiments were conducted from: April - July (trial 1), June- September (trial 2) and September - December (trial 3), respectively. The trials were conducted at the University of Hawaii Poamoho Research Station (elevation 265 m) on Oahu, Hawaii. This area was chosen because there were no known banana plants in the vicinity and thus this would limit the likelihood of infection occurring from external sources. All banana plantlets used for the field experiments were micro-propagated from disease-free banana plants. For trial 1, 140 banana plants [cv. Williams; AAA genome (derived exclusively from Musa acuminata)] were transplanted on April 13 in a 26 x 24 m plot. The spacing between rows and plants were 2.4 and 1.8 m, respectively. For trials 2 and 3, 160 and 144 healthy transplants were planted on June 15 and September 16, respectively. The plot sizes for trials 2 and 3 were 34 x 27 m and 34 x 30 m, respectively, and spacing between rows and plants were 2.4 and 2.1 m for each plot.

Plants at the five to six leaf stages were inoculated in each trial. For trial 1, 32 test plants were randomly selected and subjected to one of two treatments: i) virus (inoculated with viruliferous aphids) or ii) non-treated control (no aphids). Groups of five apterous aphids were removed from a single BBTV infected source plant on which they were reared, using a fine haired brush and placed in the ‘throat’ of the pseudostem at the 3rd youngest fully expanded leaf. Five aphids were used to increase the likelihood of successful virus transmission (Hu et al., 1996). Sixteen plants were inoculated with viruliferous aphids and another 16 plants were randomly chosen as non-treated controls. For trials 2 and 3, 10 aphids were collected from infected source plants in the laboratory and placed in the throat of the pseudostem at the 3rd and 4th youngest fully expanded leaves, respectively, of 24 plants (total of 10 aphids per plant). An additional 24 test plants were used as non-treated controls. Immediately after placing the aphids on the plants, a sleeve cage constructed of a 36 mesh cm-1 transparent fabric (Super Poly Organza, Hyman Hendler and Sons, Los Angeles, CA) was carefully placed over all the test plants. The sleeve cages were used to protect aphids from natural enemies. After five days, the sleeve cages were removed and the plants sprayed with imidicloprid (Provado 1.6F®, Bayer CropScience Inc.) at a rate of 0.7ml/L of H2O using a hand-pumped backpack sprayer.

Plant growth and chlorophyll measurements for all trials were initiated at 5 days after termination of the aphid inoculation period and taken every 5 days for trial 1 and every 10 days for trials 2 and 3, respectively. The measurements were terminated at 90 days after insect inoculation. Plant height, pseudostem diameter, and leaf area and emergence were recorded on each sampling occasion. Leaf area was estimated by measuring the leaf length and maximum width and multiplying the results by a conversion factor of 0.83 (length x width x 0.83) (Robinson & Neil, 1985). The number of leaves was counted for all test plants at each date. Leaf production rates were determined as measured by Turner (1971). As such, a leaf was regarded as emerged if the ventral surface of the midrib was fully exposed and at least half the leaf was unfurled. Plant height was measured as the distance from the ground to the fork created by the petioles of the uppermost fully emerged leaf (Smith et al., 2000). The diameter of the pseudostem was measured at the area immediately below the fifth most recently fully expanded leaf.

The relative chlorophyll content of banana leaves was determined with a Minolta SPAD-502 ® Chlorophyll Meter (Minolta Corporation, Ramsey, NJ, USA). The SPAD meter determines the greenness of the leaf and the interaction of thylakoid chlorophyll with incident light (Jifon et al., 2005). Six readings representative of the entire leaf length were taken from the edge of the most recently matured fully unfolded leaf. The average of the six readings was recorded from each test plant. Afterwards, three 6.3 mm diameter discs representing the entire lamina length were punched along the same leaf area where the SPAD readings were taken and used to determine chlorophylls a and b content. Individual leaf discs collected for chlorophyll extraction were immediately placed in 1.5 ml micro-centrifuge tubes containing dimethyl sulphoxide (DMSO), and placed in a brown paper bag. A standard chlorophyll extraction protocol was used (Richardson et al., 2002). The absorbance was measured with a Genesys 5 Spectrophotometer (Spectronic Unicam New York) at 663 and 645 nm (Richardson et al., 2002). The absorption spectra of Chlorophyll (between 600-680 nm) extracted in 90% acetone or DMSO are very similar (Hiscox & Israelstam, 1979), allowing Arnon’s equations (Arnon, 1949) to be used to calculate the Chlorophyll content.

All test plants were inspected for disease [symptoms] at 5 day intervals, commencing 10 days after aphid inoculation (e.g., margin of leaves faintly chlorotic) until 90 days after planting. Dates that banana plants were first observed displaying symptoms were recorded. For each trial, PCR was used to test plants for BBTV 10 days after aphid inoculation. Another, sample was taken 5 days later and conducted thereafter at 15 day intervals for trials 1 and 2, and 20 day intervals for trial 3, respectively. A 6.3 mm diameter cork borer was used to obtain a disc sample near the mid-vein of the most recently developed fully open leaf. A fire-torch was used to sterilize the borer in between samples; preliminary work showed no transfer of virus from sample to sample using this approach. Plant tissue was ground with sterile plastic mortars and the DNA extracted from the leaf samples with a Qiagen DNeasy kit following manufacturer's instructions (Qiagen Inc., Valencia, CA).

Spread of an introduced vector-borne banana virus in Hawaii
Samples of BBTV from symptomatic banana plants from all main Hawaiian Islands were collected between October and December, 2005. The only exception was Lanai where BBTV has not yet been reported. Samples were freshly processed or maintained frozen in -80oC until DNA extraction. We extracted viral DNA using a Qiagen DNeasy extraction kit (Qiagen Inc., Valencia, CA) following manufacturer’s instructions. Here we provide a brief description of the inter-island spread of BBTV in Hawaii, based on information provided by the Hawaii Department of Agriculture for historical data (unpublished surveys, prepared by Larry Nakahara, Hawaii Department of Agriculture) and personal observations on the current status of the disease (Almeida, personal observations).

To ascertain the origin of the Hawaiian BBTV invasion, we constructed a phylogeny based on DNA-1 sequences (coding for replication initiation protein) with sequences from Hawaiian and worldwide BBTV infections. In order to more thoroughly test the appropriateness of CCV as an outgroup, we also investigated the phylogenetic relationships between the six recognized viruses in Nanoviridae.

To infer the intra-archipelago source pools of BBTV spread across the Hawaiian Islands we mapped the observed geographic data for Hawaiian BBTV distribution on to the single dated phylogeny (with branch lengths estimated as a function of time). We conducted ancestral geographic state reconstructions under the same likelihood method as above using Mesquite v1.11. Current geographic field data of island infections was coded as a six-state, unordered state matrix (Kauai, Oahu Molokai, Maui, West Hawaii, and East Hawaii).

The historical infection dynamics of BBTV spread in the Hawaii archipelago were estimated simultaneously with the dated phylogenetic reconstruction of the Hawaiian BBTV infection (above). Effective numbers of infections (Ne) in years before present (ybp) were projected under a five-group (m = 5) Bayesian skyline plot estimation (Drummond et al 2005). The Skyline plot was estimated under the same likelihood model of base pair substitution (HKY) and initial priors as from above. Hawaiian BBTV is a unique data set for historical population estimation because i) it is spread over a geographically diverse region (i.e. an island archipelago), ii) it represents a single infection or population of BBTV, and iii) the data generated from five viral components drawn from a worldwide sample produces a well-supported and resolved phylogeny for regional infections (several well supported clades; Hawaii, Maui, et al.). Although a prior substitution rate for the Hawaiian infection is not known, strong evidence for the initial Hawaiian BBTV infection in 1989 (Conant 1992) can be used to estimate mean substitution rate. The substitution rate was not fixed, and was allowed to sample the posterior distribution reflecting absent prior knowledge of a substitution rate for BBTV.

We conducted a sequential transfer experiment to estimate the rate of mutations in BBTV under controlled conditions in relation to the number of vector transmission events. For this purpose, we allowed single aphid vectors (P. nigronervosa) to acquire BBTV from symptomatic plants for 24 hours and transferred these insects to healthy tissue-cultured banana plantlets for a 24-hour inoculation access period. This process was initiated with one individual symptomatic plant, which generated 14 parallel BBTV-infection lineages that were sequentially transferred, as described above, for 10 generations (i.e. 10 new plants were sequentially infected by individual aphids, one aphid per plant). Because under greenhouse conditions plants became symptomatic with BBTV within approximately one month, transfers were made every 4-5 weeks after infection of a new plant generation. The experiment took one year to complete. Aphids were reared on healthy banana plants and randomly collected from our colony for transmission tests. Details on general insect and plant maintenance, experimental aphid infection protocols, and greenhouse conditions have been described elsewhere (Robson et al. 2007). We kept tissue and extracted DNA samples from all lineages and passages. After 10 generations, we sequenced the same 5 DNA fragments used for the phylogeographic analyses for the original infected plant and all the descendents (last generation) of the 14 lineages.

Effect of Temperature, Vector Life Stage, and Plant Access Period on Transmission of Banana bunchy top virus to Banana

To determine if BBTV transmission requires a latent period within aphids we used leaf cuttings as BBTV sources as described above. Adult aphids (120 per plate) were transferred onto these leaves for a 12-hour AAP. To determine the latent period threshold for BBTV transmission we used IAP (8, 16, 24, 32, and 40 hours) with 10 plants per period, by transferring groups of 5 insects immediately after the 12 hour AAP to test plants. Every 8 hours, we randomly selected ten plants and removed the aphids from them. Two replicates of this experiment were conducted. We used a generalized linear model with binomial error distribution to analyze the effect of “insect latent period threshold” on “transmission rate to plants” (3). There was no statistical difference between the two runs, therefore we pooled them for all analyses.

Effect of temperature and vector life stage on transmission: We compared the transmission efficiency of adults with third instar P. nigronervosa at three different temperatures (20, 25 and 30˚C). Leaf cuttings from BBTV positive plants were used as virus source for insects. We transferred 50 adult and third instar aphids to cuttings (three cuttings per treatment) for virus acquisition. No temperature conditioning period was used for the insects. One Petri dish with adults and another with nymphs were placed in chambers set at 20, 25 and 30˚C for a 24-hour AAP. After the AAP we individually transferred the insects onto test plants. We did all insect transfers at room temperature. Test plants were covered with aphid-proof cages and returned to the same chamber for a 24-hour IAP. Twenty test plants were used per group (10 adults and 10 nymphs) in each chamber. Following the IAP we treated all plants with insecticide and placed them in the insect-free room for symptom development and PCR detection tests. This experiment was repeated twice, with each repetition treated as a statistical replicate.

Experiments were conducted to identify the relative importance of temperature on BBTV acquisition and inoculation efficiency by adult aphids. To determine the effect of temperature on acquisition, we varied the temperature during the AAP while keeping it constant for the IAP. We did the inverse to determine the impact of temperature on inoculation (i.e. AAP temperature constant while IAP temperature was variable). To study the effect of temperature on acquisition we placed adults on BBTV-infected leaf cuttings for virus acquisition. There was no insect temperature acclimation period prior to plant access period. We kept cuttings in growth chambers set at 20, 25 and 30oC for a 24-hour AAP, after which we transferred the insects onto test plants (1 aphid per plant / 10 plants per temperature during acquisition) for a 24-hour IAP in a 25oC chamber. Similarly, to determine the effect of temperature on inoculation efficiency we placed aphids on BBTV-positive cuttings for a 24-hour AAP at 25oC, after which we transferred aphids onto healthy plants at 20, 25 and 30oC for a 24-hour IAP (total of 10 individuals per treatment). This experiment was repeated twice, with each repetition treated as a statistical replicate.

Effect of plant access time on transmission efficiency: We determined BBTV transmission efficiency by individual P. nigronervosa adults at 25oC by varying the AAP, while keeping IAP constant, or by using a constant AAP followed by variable IAP. The constant AAP and IAP were 24 hours. We used 0.5, 1, 2, 6, 12, 18, and 24 hours as the variable IAP and AAP. Ten adult aphids (1 per plant) were tested per plant access period. BBTV-positive leaf cuttings were used for virus acquisition and healthy plantlets for inoculation. We analyzed the results with linear regressions to estimate the relationship between plant access time and the proportion of plants infected.

We placed individual adult aphids onto banana leaf midrib cuttings in petri dishes as described above. Trays with petri dishes/aphids were placed into controlled environment growth chambers at 20, 25, and 30ºC with a 12-h photoperiod. We replaced leaf cuttings every 3 to 4 d. Plates were observed daily for the presence of offspring. When nymphs were first found, we removed the adults and left one offspring in each dish. We observed aphids individually on a daily basis until they reached adulthood to determine development time at the different temperatures. After adulthood was reached, we recorded the number of offspring present daily. We constructed complete life tables according to Carey (1993) with the data collected. Intrinsic rate of increase (r), the number of offspring produced per unit time was assessed.

We placed individual fourth-instar aphids on potted banana plantlets at the five to six leaf stage that were kept in controlled environment growth chambers at three different temperatures: 20, 25, and 30ºC. We kept plants in 8.9-cm square pots that were covered with cloth mesh cages. We used rubber bands around the cage and pot to prevent aphids from escaping. For each treatment, we placed 16 banana plantlets onto a tray that was placed onto a shelf in the growth chamber. Plants were watered as needed from below by pouring water into the tray. We randomly chose four plants from each treatment to count aphid numbers on a weekly basis for 1 mo, sampling without replacement.

Research results and discussion:

Monitoring of pest densities allows for informed decision making regarding the judicious use of pesticides. Prior to the start of this project, there were no sampling plans available for monitoring population densities of P. nigronervosa on banana. Thus, field surveys were conducted to develop a sampling plan for this pest. Based on these surveys, a sequential binomial sampling plan has been developed. Further, it was determined that the within-plant distribution of P. nigronervosa is an important factor to consider when sampling for this pest. Specifically, aphids were found more frequently near the base of banana plants, followed by the newest unfurled leaf at the top of the plant. Aphids were least likely to be located on leaves in between the top and bottom of the plant. In the past it was widely accepted among banana growers in Hawaii that banana aphid occurs preferentially on the cigar leaf (newest unfurled leaf). During this study, it was determined that sampling the cigar leaf for aphid presence is not a reliable technique because in 50% of the cases this provides a false negative for their presence This sampling plan is currently assisting in the development of sustainable management practices for banana production and the results from this study was recently published in a manuscript entitled “Within-Plant Distribution and Binomial Sampling of Pentalonia nigronervosa (Hemiptera: Aphididae) on Banana” Journal of Economic Entomology, 99(6): 2185-2190 authored by Robson et al., 2006. Future research needs include evaluating this sampling plan with insecticides and cultural management tactics such as roughing for the further development of disease management strategies.

In an effort to learn more about the distribution of P. nigronervosa and to help disseminate information to growers with regards to BBTV management within banana mats, several farm surveys were conducted. Banana mats usually consist of at least one mature or nearly mature banana plant and some smaller plants or suckers. Banana suckers are vertical shoots that develop from the base of a banana pseudostem. Twenty nine banana plantings were visited throughout the state of Hawaii and 26 of these farms were surveyed for aphids, nematodes, and BBTV. Afterwards, each farm was given information on how to best manage the banana aphid, BBTV and nematodes. These visits included commercial growers, banana hobbyist, and individuals whose banana plantings were not their major sources of farm income. In addition, to aphids other banana pests of concerns such as corm weevils, nematodes, and black leaf streak disease were addressed during or after each farm visit. Our preliminary findings from the aphid surveys suggest that P. nigronervosa populations are greater on plants approximately 1.5 meters or less in height and only under conditions of high populations are high numbers found on larger plants (i.e., 3 meters or greater). Additionally, banana aphids are not found on the fruit or flower of banana plants. This would suggest that sampling plants 1.5 meters in height or less would be feasible for scouting banana aphids. This data is currently being analyzed and findings will be used to further advise growers on how to scout their fields for P. nigronervosa and which size plants should be targeted when the economic threshold has been reached. During the surveys, it was noted that many fields were infested with BBTV and nematode pests. This has led us to investigate whether banana plants physiologically stressed by nematodes express BBTV symptoms differently than plants with out nematodes.

Further, it was suggested that to help control the banana aphid, farmers should also target ants. The belief is that ants are tending aphids and protecting them from natural enemies. However, findings during the survey suggest that ants are not providing any significant benefits to ants, and in many instances ants were observed attending other herbivores such as mealybug and scale insects. Additionally, ants can be readily found in high numbers in the absent of ants. This suggests that it is not necessary for banana growers to adopt practices aimed at suppressing ants. Ultimately, these practices could lead to additional production costs with no benefits.

Few studies have been conducted to learn more about the biology and ecology of P. nigronervosa. Thus, studies were carried out to determine the effect of temperature and rearing methods on P. nigronervosa biology. Data on population growth, longevity, and fecundity of P. nigronervosa at different temperatures are useful for developing prediction models for aphid population dynamic under field conditions. During these studies, aphids were evaluated at three temperatures (20, 25, and 30º C). These temperatures represent the temperature range of banana growing areas in the Hawaiian Islands. Banana aphids were reared on six different types of banana leaf cuttings. i) mature leaf >1 m in length, ii) young leaf < 0.5m in length, iii) leaf petiole, iv) leaf midrib, (v) symptomatic BBTV leaf, and vi) cigar leaf (newest unfurled leaf). It was found that the banana aphid performed better when not confined on plantlets, followed by leaf midrib cuttings. Further, their intrinsic rate of increase (r), net reproductive rate (Ro), doubling time (DT), nymphal mortality, and mean offspring per female all showed maximal rates at 25ºC. The r was greater when aphids were reared on intact banana plantlets than on plant cuttings. The results showed that it is important to conduct whole plant experiments when evaluating banana aphid growth features because various growth parameters may be underestimated if various plant cuttings are used. This further implicates the importance of comparing insect rearing methods for studies such as life tables.

Results of this experiment may help predict at what temperatures banana aphid populations will build to greater numbers, assisting in the development of management practices aimed to control the banana aphid and BBTD. Future studies, need to examine the population thresholds that induce the production of alate (winged) banana aphids because the alates are most responsible for spreading the virus. Additional, studies are also needed for determining biotic and abiotic factors affecting banana aphid population growth in the field. The findings from this study were justly published under the title “Biology of Pentalonia nigronervosa (Hemiptera: Aphididae) on Banana Using Different Rearing Methods” Environmental Entomology 36(1): 46-52 authored by Robson et al. 2007.

There are ongoing studies to learn more about the biology and ecology of the banana aphid but much of the attention is now focused on how this relates to their ability to transmit BBTV to healthy banana plants. Although, previous research (Robson et al., 2007) indicates that various growth parameters of the banana aphid is significantly reduced on plant cuttings compared to whole plants, we learned that their efficiency in obtaining and transmitting BBTV to healthy banana plants is similar whether they feed on whole plant or leaf cuttings infected with BBTV, respectively (Figure 1).

In controlled experiments, it was also found that BBTV is transmitted by the banana aphid between 20 and 28 hours after feeding on an infected plant (acquisition) including the 12 hour acquisition access time. In two trials, the transmission efficiency for the 28 hour treatment period was 50 and 60%, respectively. Their transmission efficiency for the 36 hour time period was 90 and 100% for trials 1 and 2, respectively (Table 1).

The minimum latent period (i.e., length of time after a plant has been inoculated with a virus that an aphid can acquire and transmit the virus from that plant) of BBTV within banana plants was investigated. The inoculation efficiency was 0 and approximately 75% at the 15 and 20 day test periods, respectively (Figure 2). This indicates that the minimum latent period is somewhere between 15 and 20 days following virus inoculation. These studies are among a few that have been conducted to examine BBTV’s transmission efficiency.

Data on the ability of the banana aphids to transmit BBTV at different temperatures are useful for understanding their transmission biology. Banana aphids were evaluated at three temperatures (20, 25, and 30º C) for their ability to transmit BBTV to healthy banana plants. Additional, studies were conducted to compare the efficiency of adult and nymphs in transmitting BBTV. During these investigations, it was determined that adults can transmit the virus at all three temperatures but they have low efficiency at 20ºC. Nymphs did not do as well compared to adults and could not transmit BBTV to healthy plants when exposed to 20ºC (Figure 3). It was also noted that banana aphids were more settled on the leaves at 25 compared to 30ºC.

An effective management strategy for BBTV is dependent on rapid detection of symptomatic plants so that potential source plants of BBTV can be destroyed promptly.
Thus, field studies were also conducted in Oahu, Hawaii to identify features of Banana bunchy top disease (BBTD) that could be used as pre-symptomatic indicators of BBTV infection. The growth and morphology of banana plants infected with BBTV and healthy controls were investigated. The time interval between aphid inoculation of BBTV and the initial appearance of visual disease symptoms (i.e. incubation period) was also determined. Plants infected with BBTV showed a significant reduction in petiole morphology, plant canopy and height, leaf area, pseudostem diameter and chlorophyll content compared with control plants. Growth differences between virus infected and control plants were not observed until 40 to 50 days after the plants were inoculated with viruliferous aphids. Other growth parameters such as petiole width and leaf production were not statistically different between infected and control plants. The results show that banana growth parameters may not be suitable as pre-symptomatic indicators of BBTV infection and that PCR assays can provide earlier detection (5 to 10 days in advance) of BBTV in banana plants compared to visual symptoms. It was also found that the initial appearance of observable BBTV symptoms ranged from 25 to 85 days after viruliferous aphid inoculation (Figure 4). Findings from this study are currently on line and in press in the Annals of Applied Biology. The title of the article is “Effects of Banana bunchy top virus infection on morphology and growth characteristics of banana”.

To learn more about the epidemiology of BBTV, GPS units were used to track the movement pattern of the virus in two commercial banana orchards. Monthly data from these farms were initiated in January 2005 and completed in January 2007. Farm 1 used known diseased-free plants (micro-propagated banana plants) for planting material and farm 2 used banana suckers removed from various fields within the farm as planting material. From the data collected from farm 1, it was determined that the virus movement was influenced by wind direction and that the spread pattern was clustered. Additionally, it was found that the virus moved approximately 30 meters per month (Figure 5). Data from farm 2 indicates that the spread pattern is random and further suggests that field to field spread of BBTV from farm 2 is mostly influenced by the unintentional planting of infected banana suckers throughout the farm.

We have established a small field of known disease-free banana plantlets on farm 2 and are now monitoring the spread of BBTV in that field. An additional 1 acre field was established at the Poamoho research station and a midsize farm in Kunia, Oahu and in May 2008 a final site was established on an organic banana farm in Hawi, Big Island. This farm and others in the area of Hawi have a history of BBTV. It has been suggested to growers in Hawaii that if they find an infected banana plant, they should destroy banana plants in neighboring mats to prevent virus spread. However, our data thus far suggest that if growers readily check and destroy infected plants the probability that neighboring plants are infected is significantly reduced and do not warrant destroying them.

A survey of 50 banana suckers was conducted just after they were transplanted at a major banana producing farm on Oahu. All suckers looked healthy and no obvious BBTV symptoms were present. It was uncovered that 100% of the suckers contained banana aphids, 92% were infested with winged banana aphids which are the morph most responsible for virus spread, and using PCR methodology it was discovered that 20% of the plants contained aphid carriers of BBTV at time of transplanting. Since some plants may take longer to test positive for the virus this is considered a conservative estimate of the percentage of suckers containing aphids with BBTV. Since these findings the grower started purchasing known clean banana plantlets develop through tissue culture.

Thus, we are continuously promoting the use of known disease-free planting material (tissue culture banana plantlets) to banana growers so that they can avoid the unintentional planting of infected banana suckers throughout their farms. We use data such as mentioned in the latter paragraph to stress the potential risks of using banana suckers as replant material especially if they are in areas where BBTV is prevalent. Workshops, interviews, press releases, and onsite farm visits are ongoing to familiarize banana growers with tissue-cultured banana plants and discuss the benefits of using these plantlets as part of an integrated disease management program. We have noticed a significant jump in the number of growers and banana hobbyist interested in using tissue-cultured banana plants in their gardens and farms. A banana tissue culture facility has been developed on UH Manoa campus to insure that BBTV-free plantlets are readily available to banana growers and BBTV banana plantlets are currently being distributed to commercial growers, banana hobbyist and home gardeners.

In indition to using GPS to monitor BBTV spread, molecular data (partial sequences of BBTV's DNA genome) are being used to address specific questions regarding the introduction and spread of this pathogen in Hawaii. Results are summarized in the following three figures.

Figure 6 illustrates the genetic placement of Hawaii BBTV isolates from different islands in relation to BBTV isolates from other regions of the world. It is important to note that Hawaii isolates form a monophyletic clade, suggesting there was only one introduction of this pathogen into the State of Hawaii. Although Hawaii's BBTV could be grouped with isolates from the South Pacific, its original source could not be pin pointed because there are no BBTV sequences deposited in databases for comparison.

Figure 7 shows the placement of isolates collected from different Hawaiian islands. Interesting enough isolates from Maui and Hawaii (Big Island) form monophyletic clades, suggesting only one introduction into those islands. On the contrary, Kauai isolates are located at different branches in the tree, suggesting multiple introductions of BBTV were made into that island. Oahu isolates are present throughout the tree, suggesting that it served as the original BBTV source area for infections into other Hawaiian islands.

Figure 8 illustrates BBTV introduction and spread hypothesis and summarizes the results obtain with the use of molecular tools. It shows that BBTV after being introduced into Oahu in 1989 (one introduction), it then spread to other islands, once into Maui and Hawaii (Big Island), respectively, and multiple times into Kauai.

Much of the data from this molecular work suggest that the spread of BBTV throughout Hawaii was greatly influenced by the movement of infected plant material throughout the islands. Similar, we have determined that the within farm spread of BBTV may be more influenced by the planting of infected banana suckers than aphid vectors. This further suggests the importance of using known disease-free banana plants as replant material.

Once a banana plant has been diagnosed as being infected with BBTV, the general recommendation is that the plant be destroyed as soon as possible to prevent it from serving as a source for continuous virus spread. The most popular method being used to destroy infected banana plants is injecting them with a bananacide (i.e., herbicides that can be used to kill banana plants). However, it was not known how long banana plants remain virulent after a bananacide injection. Four on farm field experiments were conducted to investigate this question. It was discovered that banana plants can remain virulent up to six weeks after being injected with a bananacide at the highest label recommended rate and that aphids are capable of acquiring the virus from plants up to 6 weeks after injections. Similarly, it was determined that aphids feeding on plants 6 week after injection can transmit BBTV to healthy banana plants. Thus growers are beginning to treat their plants with an insecticide after injection. The importance of this finding is that many growers believed that an infected plant was no longer a threat after being injected with a bananacide. Additional, studies are needed to determine non-chemical/organically acceptable methods for destroying infected banana plants and determining if injecting plants with a bananacide causes greater production of winged morphs. Future plans are underway to determine how BBTV titer levels changes in a plant following a bananacide injection.

Several field experiments were conducted to determine if the two most economically important banana cultivars [apple/dwarf Brazilian (AAB Group) and Williams (AAA Group)] grown in Hawaii differed in their susceptibility to BBTV. Results of these studies indicated that several growth (e.g., plant height, canopy, leaf area, etc.) and physiological features (chlorophyll and moisture levels) of both cultivars were similarly impacted by BBTV. The chlorophyll content was significantly less in plants of both cultivars infected with BBTV compared with control plants (Figure 9). However, Williams appeared to have a wider incubation range than apple (Figure 10). Despite morphological and physiological similarities, significantly fewer dwarf Brazilian became infected compared to the Williams banana (Table 2). These finding differed from results obtained from a laboratory study in which similar numbers of apple and Williams banana plants became infected (Table 3). Also, on average 1 additional leaf were produced prior to the appearance of symptoms in apple compared to Williams banana plants during both laboratory and field trials. It is the generally believed by commercial banana growers in Hawaii that apple is more resistant to BBTV than Williams. Findings from the field investigation added some credence to this belief. This study provides evidence that different banana cultivars vary in their susceptibility to BBTV. Because, there are no known banana cultivars resistant or tolerant to BBTV, the use of less susceptible cultivars may be an alternative management option that can be readily incorporated into an overall BBTV management plan. However, results from this experiment dispelled the myth that apple is more tolerant of BBTV or mask its symptoms better than Williams banana. More research is needed to determine the factor(s) responsible for lower virus incidence in apple bananas. Further, much of the results obtained here continue to support our earlier belief that the efficiency of P. nigronervosa to transmit BBTV is significantly reduced in a field environment. Thus, vector transmission efficiency data on BBTV obtained from laboratory experiment should not be used to predict BBTV transmission probability in the field because of its potential to significantly overestimate BBTV infection rates. Further research is needed to evaluate other cultivars for their susceptibility to BBTV.

Recently, imidacloprid a systemic insecticide was registered for managing P. nigronervosa in bananas. A field study was conducted to determine how long after application does imidacloprid residual activity remain effective in controlling P. nigronervosa and to compare the residual activity of imidacloprid with two rates below the recommended label rate. Additional data was taken on its impact on aphid reproduction. The data from this study are presented in Figures 11 and 12. It was found that the effectiveness of Provado 1.6F ® is short lived especially compared to laboratory findings. At the label recommended rate, survival of aphids increases significantly beyond 10 days after application (Fig. 11). Further, the numbers of aphid offsprings produced become similar to the no spray (check) treatment between 5 and 10 days after spray application. Current plans are to determine how long after applications is it effective in preventing viruliferous aphids from infecting healthy banana plants.

We are also monitoring the natural spread of BBTV from fields grown with tissue cultured plants. In each instance the plantings were conducted in areas with a history of BBTV. The primary research objective is to obtain a better understanding of how BBTV spreads. The main outreach objective is to demonstrate that areas with history of high occurrences of BBTV can be rehabilitated if individuals aggressively scout for and destroy infected plants and use known BBTV-free plants as replant material. Figure 13 shows a banana plot located at the University of Hawaii at Manoa Poamoho Research Station at 16 months after planting.

References

Carey, J. R. 1993. Applied demography for biologists with special emphasis on insects. Oxford University Press,New York.

Conant, P. (1992). Banana bunchy top disease, a new threat to banana cultivation in Hawaii. Proceedings of the Hawaiian Entomological Society, 31, 91-95.

Constantinides, L.N., McHugh Jr., J.J. (2003). Pest Management Strategic Plan for Banana Production in Hawaii. Pearl City Urban Garden Center, Workshop Summary, pg 1-71.

Dale, J.L.,Harding, R.M. (1998). Banana bunchy top disease: current and future stratified for control. Plant Virus Disease Control. A. Hadidi, Khetarpal, R. K. and Koganezawa, H. St. Paul, Minnesota, APS Press, pgs., 659-669.

Ferreira, S., 1991. The status of moko and bunchy top diseases in Hawaii. Res. Ext. Ser., Coll. Trop. Agric. Hum. Resour., Univ. Hawaii Coop. Ext. Ser., Honolulu, HI. The Service 124: 180-183.

Hiscox, J.D., and Israelstam, G.E. (1979). A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany, 57, 1332-1334.

Hu, J.S., Wang, M., Sether, D., Xie, W., Leonhardt, K.W., 1996. Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa), Annals of Applied Biology, 128, 55-64.

Jifon, J.L., Syvertsen, J.P., Whaley, E. (2005). Growth environment and leaf anatomy affect nondestructive estimates of chlorophyll and nitrogen in citrus sp. leaves. Journal of American Society of Horticultural Science, 130, 152-158.

Magee, C.J.P. (1927). Investigation on the bunchy top disease of the banana. Council for Scientific and Industrial Research, Bulletin 30.

National Agricultural Statistics Service. 2005. Statistics of Hawaii Agriculture: Hawaii Bananas. U.S. Department of Agriculture, Honolulu, HI.

Richardson, A.D., Duigan, S.P., Berlyn, G. P. (2002). An evaluation of noninvasive methods to estimate foliar chlorophyll content. New Phytol. 153, 185-194.

Robinson, J. C. 1996. Bananas and plantains. CAB International, Wallingford, United Kingdom.

Robson, J. D., Wright, M. G., and Almeida, R. P. P. 2007. Biology of Pentalonia nigronervosa (Hemiptera, Aphididae) on banana using different rearing methods. Environ. Entomol. 36:46-52.

Romoser, W. S., and J.G.J. Stoffolano. 1998. The science of entomology. WCB/McGraw-Hill, Boston, MA.

van Hoof, H. A. 1962. Observations on aphid flights in Surinam. Entomol. Exp. Appl. 5: 239 - 243.

Young, C. L., and M. G. Wright. 2005. Seasonal and spatial distribution of banana aphid, Pentalonia nigronervosa (Hemiptera: Aphididae), in banana plantations on Oahu. Proc. Hawaiian Entomol. Soc. 37: 73-80

Research conclusions:

Our results are being disseminated to banana stakeholders through workshops, farm site visits, collaborative research projects, websites, conferences and email action groups Each year, we highlight relevant findings at the local banana conference and from stakeholders comments and questions determine additional research needs. In the pass, Hawaii growers were given limited information regarding the virus and vector and thus relied on their own guesswork on how to best manage this virus. Thus, one of the leading constraints to greater adoption of IPM strategies for BBTV management in Hawaii was the availability of trustworthy information. Through our collaborative research and outreach efforts, we have been able to give stakeholders creditable information on the virus and its associated vector and have thus removed some of the questionable myths regarding Banana bunchy top virus.

We have also built a diverse team of collaborators that include administrators, extension agents/personnel, researchers, farmer cooperators, non-profit agencies (e.g., Hawaii Banana Industry Association, Oahu Banana Growers Association, Maui Invasive Species Committee), students, and service providers. This team is also multi-disciplinary and includes Plant Pathologists, Nematologists and Entomologists.

Thirty two banana plantings have been visited throughout the state of Hawaii and 26 of these farms and have been survey for aphids, nematodes, and BBTV. Each farm was given information on how to best manage the banana aphid and BBTV. These visits included commercial growers, banana hobbyist, and individuals whose banana plantings were not their major sources of farm income. In addition, to BBTV other banana pest of concerns such as corm weevils, nematodes, and black leaf streak disease were addressed during or after each farm visit.

Since the start of the project, growers have gained a better understanding of the virus and have changed their production practices in an effort to better manage the disease. We now expect to see a reversal in banana acreage lost because of this intrepid virus. In addition, to accomplishing the original objectives of the project, we instituted additional goals that will substantially benefit the banana industry in Hawaii. Further, two graduate students received their Master of Science degree in May 2006 and August 2007 on research work related to this project. A third graduate student who contributed to various aspects of the project completed his Master of Science degree in December 2007. An undergraduate who directed research project allowed her to participate on the project, will graduate in Fall 2008.

Research from this project has been highlighted in a USDA-CSREES movie and collaborators of this project have contributed to the design of a BBTV awareness poster, Hawaii Department of Agriculture BBTV pamphlets, a video on BBTV in Hawaii (http://www.ctahr.hawaii.edu/bbtd/). Our goal to slow the spread of BBTV was highlighted on the front page of the University of Hawaii main web page “http: //www.hawaii.edu/” and the front page story of the December issue of the College of Tropical Agriculture and Human Resources (CTAHR) news magazine “http://www.ctahr.hawaii.edu/acad/Research/ResearchNews.html”. Interviews and press releases regarding BBTV were conducted with several local newspapers on the islands of Oahu, Lanai, and Hawaii. An interview was also conducted with Hawaii Public Radio. Five papers have been published in peer review journals, three additional has been submitted for publication and 2 are being prepared for submittal. Slide presentations highlighting some of our work are posted online “http://www2.hawaii.edu/~snelson/HBIA/”. Thirty onsite farm visits have been conducted on 5 Hawaiian Islands. Three demonstration plots have been set up at three collaborative farm sites. Additionally, we have communicated with ~ 200 persons through phone and email conversations with regards to BBTV management.

Results from molecular laboratory procedures show that the main driving factor behind the Hawaiian BBTV epidemic was most likely the transportation of contaminated plant material among islands from Oahu. The total number of established inter-island invasions observed (n=6) in less than 20 years highlights the difficulty in controlling the movement of infectious hosts if those are cultivated plants that remain asymptomatic for many weeks after infection.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

Graduate Degrees:

Ms. Mandy D. Anhalt "Transmission Biology of Banana Bunchy Top virus to Banana by Pentalonia Nigronervosa (Hemiptera: Aphididae)". MS Thesis, University of Hawaii at Manoa. May 2007.

Ms. Jacqueline Robson "Biology and Ecology of Pentalonia Nigronervosa Coq. in Hawaii and Aspects of its Chemical Control with Imidacloprid". Ms Thesis, University of Hawaii at Manoa. May 2006.

Publications:

Robson, J.D., Wright, M.G. and Almeida, R.P.P. 2006. Within-plant distribution and binomial sampling of /Pentalonia nigronervosa/(Hemiptera, Aphididae) on banana. Journal of Economic Entomology, 99(6): 2185-2190.

Robson, J.D., Wright, M.G. and Almeida, R.P.P. 2007. Biology of Pentalonia nigronervosa/(Hemiptera, Aphididae) on banana using different rearing methods. Environmental Entomology, 36(1): 46-52.

Robson, J.D., Wright, M.G. and Almeida, R.P.P. 2007. Effect of imidacloprid foliar application on banana aphid /Pentalonia nigronervosa/ (Hemiptera, Aphididae) survival. - New Zealand Journal of Crop and Horticultural Science 35: 415-422.

Hooks, C. R. R., Wright, M.G., Kabasawa, D. S., Manandhar, R., Almeida, R. P. P. Effect of Banana bunchy top virus infection on morphology and growth characteristics of banana. - Annals of Applied Biology, In Press.

Anhalt, M.D. and Rodrigo P.P. Almeida 2008. Effect of Temperature, Vector Life Stage and Plant Access Period on Transmission of Banana bunchy top virus to Banana. Phytopathology, In Press.

Almeida, R. P.P. G.M. Bennett, M. D. Anhalt, C.W. Tsai and P. O’Grady. 2008 Spread of an Introduced Vector-Borne Banana Virus in Hawaii. Molecular Ecology Submitted.

Hooks, C.R.R., R. Manandhar, E. P. Perez, K.-H. Wang, and R.P.P. Almeida. Comparative susceptibility of banana cultivars dwarf Brazilian and Williams to Banana bunchy top virus. Plant Disease Submitted

Hooks, C. R.R., S. Fukuda, R. Manandhar, E. P. Perez, K.-H. Wang, and R.P.P. Almeida. The virulence of Banana bunchy top virus in banana plants after injection with a bananacide. In preparation

Wang, K.-H. and Hooks, C. R. R. Survey of plant parasitic and beneficial nematodes associated with banana agroecosystem in Hawaii. In preparation

Abstracts:

Almeida, R.P.P. 2006. Molecular epidemiology of /Banana bunchy top virus/ in Hawaii. Phytopathology 96 (Supplement): S5.

Presentations at conferences:

Wright, M.G., Almeida, R.P.P., Hooks, C.R.R. and Robson, J.D. Aphids and banana bunchy top virus in Hawaii. Invited presentation. 2005 Pacific Entomology Conference, Invasive Species Symposium.

Robson, J.D., Wright, M.G. and Almeida, R.P.P. Biology and ecology of Pentalonia nigronervosa Coq. (Hemiptera: Aphididae) in Hawaii. Annual Meeting of the Entomological Society of America. December 15-18 2005. Fort Lauderdale, FL.

Almeida, R.P.P. Biology and epidemiology of banana bunchy top virus. WERA20 Regional Meeting. May 8-9 2006. Victoria, Canada.

Anhalt, M.A. and Almeida, R.P.P. Transmission of Banana bunchy top virus to banana by Pentalonia nigronervosa (Hemiptera: Aphididae). Annual Meeting of the the Entomological Society of America. December 2006, Indianapolis, IN.

Hooks, C.R.R., Wright, M.G., Kabasawa, D. S., Manandhar, R., Almeida, R P.P. The influence of Banana bunchy top virus infection on the morphology and growth of banana. 37th Annual Hawaii Banana Industry Association Conference. August 25th, 2006. Hilo, Hawaii.

Almeida, R.P.P. and Vorsino, A. Spread of Banana bunchy top in Hawaii. 37th Annual Hawaii Banana Industry Association Conference. August 25th, 2006. Hilo, Hawaii.

Anhalt, M.A and R.P.P. Almeida, Transmission biology of banana bunchy top virus: viral acquisition and latent periods in aphids and banana plants. 37th Annual Hawaii Banana Industry Association Conference. August 25th, 2006. Hilo, Hawaii.

Wright, M.G., Pest biology, sampling, management decisions and provado for the management of banana aphids. 37th Annual Hawaii Banana Industry Association Conference. August 25th, 2006. Hilo, Hawaii.

Mandy D. Anhalt, Rodrigo PP Almeida, Mark G. Wright, Cerruti RR Hooks, & Jane M. Tavares, Transmission Biology of Banana bunchy top virus by Pentalonia nigonervosa (Hemiptera: Aphididae). 38th Annual Hawaii Banana Industry Association Conference. August 2007. Oahu, Hawaii.

Cerruti RR Hooks, Steve Fukuda, Eden A. Perez, Derek Kabasawa, Mark G. Wright, Roshan Manandhar, Koon-Hui Wang, & Rodrigo PP Almeida, The virulence of Banana bunchy top virus in Banana Plants After Injection With a Bananacide. 38th Annual Hawaii Banana Industry Association Conference. August 2007. Oahu, Hawaii.

Eden A. Perez, Cerruti RR Hooks, Mark G. Wright, Mandy Anhalt, & Rodrigo PP Almeida, Integrated BBTV Management: Using a Combination of Strategies to Control the Virus and its Aphid Vector. 38th Annual Hawaii Banana Industry Association Conference. August 2007. Oahu, Hawaii.

Koon-hui Wang, Cerruti RR Hooks, & Roshan Mandahar, Preliminary Investigation of Nematodes Inhabiting Banana Fields in Hawaii and Their Management Options. 38th Annual Hawaii Banana Industry Association Conference. August 2007. Oahu, Hawaii.

Mark G. Wright, Sequential Sampling: How to Decide When to ‘Take Action’ Based on Threshold Counts. 38th Annual Hawaii Banana Industry Association Conference. August 2007. Oahu, Hawaii.

Workshops/Moderated Discussion/Public Outreach

Jari Sugano, Cerruti RR Hooks, Mandy Anhalt, Derek Kabasawa, Update of findings from current banana research. Banana growers on Oahu. May, 2006. Kaneohe Extension Office, Oahu, Hawaii.

Kabi Neupane, Cerruti RR Hooks, Eden Perez, Derek Kabasawa, Introduction to Banana Tissue Culture and Disease-Free Planting Material. July 13, 2006. Leeward Community College, Oahu, Hawaii.

Cerruti RR Hooks, Mandy Anhalt, Mark Wright, Banana workshop: Update and future direction of banana research. May, 2007. University of Hawaii at Manoa, Oahu, HI.

Jim Hollyer, Banana tissue culture plant distribution policy, moderated discussion. 38th Annual Hawaii Banana Industry Association Conference. August 2007. Oahu, Hawaii.

Cerruti RR Hooks, Mandy Anhalt, Mark Wright, Banana workshop: Update and future direction of banana research. May, 2007. University of Hawaii at Manoa, Oahu, HI.

Cerruti RR Hooks, Eden Perez, Koon-Hui Wang, Banana workshop: Use and care of tissue cultured plants as part of an integrated disease management strategy. March, 2008. Kaneohe Oahu, HI.

Cerruti RR Hooks, Eden Perez, Koon-Hui Wang, University of Hawaii at Manoa, CTAHR centennial Celebrations: Managing banana bunchy top virus. March, 2008. Pearl City, Oahu, HI.

Cerruti RR Hooks, Scot Nelson, Koon-Hui Wang, Banana workshop: A compendious overview of BBTV and its aphid vector, Pentalonia nigronervosa April, 2008. Hawi Community Center, Big Island, HI.

Press releases & interviews about BBTV and tissue cultured banana plants:

1) http://www.hawaii.edu/cgi-bin/uhmnews 20071214103107 C.R.R Hooks, K.-H. Wang
2) Kohala Mountain News, Big Island, HI, December 2007 C.R.R Hooks, K.-H. Wang
3) The Lāna’i Times, Lana’i, HI, December 2007 K.-H. Wang
4) Honolulu Star Bulletin, Oahu, HI, December 2007 C.R.R Hooks, K.-H. Wang
5) Hawaii Public Radio Station December 2007 C. R. R. Hooks

Project Outcomes

Project outcomes:

It has been suggested that BBTD will persist almost indefinitely within plantations because inspectors are unable to identify all infected plants during a single inspection, and non-symptomatic infected plants that remain between inspections may serve as a source of inoculum for virus spread. Additionally, it is not possible to look at a plant and determine whether it is virus free. Thus, growers in Hawaii using the conventional planting technique (sucker planting) in BBTV areas help increase the rate of BBTV spread by inadvertently augmenting the number of infected plants that will be overlooked during inspection periods. Further, the labor required to dig up and replant infected suckers is laborious and costly, and there is a risk that infected suckers are constantly being replaced with additional infected suckers. This constant destruction and replanting of diseases suckers adds additional production costs. Using tissue cultured plants derived from BBTV-free parent material is one way to insure that the plants are free of BBTV at planting. Additionally, these plants require much less labor and time to plant compared to using banana suckers as planting material. We cannot provide a detail economical impact of this project. However, the fact that some growers are reducing their pesticide usage as a result of our outreach efforts, suggests that there is some savings associated with reduced chemical use. The National Agricultural Statistics Service report on Hawaii banana production for the years 2007 and 2008 will provide us greater insight on the economic benefits of our program.

Farmer Adoption

Because the planting of infected plant material is a major contributor to BBTV spread, we rank our achievement in promoting the use of known disease-free banana planting material, establishing a permanent program within the University of Hawaii at Manoa to produce disease-free banana plantlets, and the distribution of known BBTV-free banana plantlets to the public among our greatest project accomplishments. There are currently 11 different banana cultivars being micro-propagated and ~ 90,000 banana plantlets have been produced. This alone has the potential to impact 160 acres of banana in Hawaii. To date ~ 200 clients (e.g., banana growers, banana hobbyist, home gardeners, researchers, etc.) have requested BBTV-free tissue-cultured banana plantlets through this program and 104 of those orders have been completed. We expect more banana stakeholders to switch from using banana suckers as planting material to using know disease-free micro-propagated plants. As this trend continues and the movement and spread of BBTV is slowed, we expect yields to become more corresponding to the amount of banana acreage in production. We estimate that ~ 50 banana growers have been reached through our program through direct farm visit, telephone and e-mail communications, workshops and conferences.

Our best recommendations to farmers and other banana stakeholders (home gardener, hobbyist, etc.) includes: 1) do not transport banana plants from one location to another, 2) do not accept banana plants from other individuals, 3) aggressively scout fields for infected plants and if found destroy them instantly, 4) only use known BBTV-free banana plants as replant material, 5) share new found trustworthy information about BBTV and its management to family, friends and other associates and 6) if there are any questions regarding BBTV please consult with an expert..

Recommendations:

Areas needing additional study

Future research needs include determining if BBTV effects the fitness of P. nigronervosa (e.g., reproduction, morph ratio, rate of development), evaluating banana plants of genomic differences for their susceptibility to BBTV and determining reason for differences in transmission efficiency between dwarf Brazilian and Williams banana cultivars, evaluating the ability of imidacloprid to prevent P. nigronervosa from acquiring and transmitting banana bunchy top virus. Determining feasible non-chemical and organically acceptable methods to destroy banana plants infected with BBTV. Locate and evaluate biological control agents that are capable of suppressing populations of P. nigronervosa. Investigate cultural management practices that can be used to help suppress important plant parasitic nematodes associated with banana plantings.

Future training needs include field day/workshop events to train home gardeners and banana hobbyist on early detection of Banana bunchy top virus. Train banana stakeholders on: 1) how to handle and care for tissue cultured banana plantlets, 2) field sanitation practices to help manage BBTV, and 3) how to scout for and use economic threshold levels to help manage P. nigronervosa.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.