Coffee berry borer (CBB), Hypothenemus hampei, is a world-wide threat to coffee (Coffea
arabica) production, causing more than 35% yield loss if harvest is delayed. Coffee is the second
most valuable commodity crop in Hawaii. Current management practices for CBB include
labor-intensive field sanitation by strip picking cherries and cleaning the ground along with multiple
expensive applications of biopesticude Beauveria bassiana. This proposed project aims
to develop sustainable pest management strategies through a conservation biological control approach by enhancing a cosmopolitan soil dwelling parasite of CBB known as entomopathogenic nematode (EPN) in the genus, Heterorhabditis. Based on ecological knowledge on EPN, these nematodes prefer higher
moisture and require UV protection, which can be provided by mulching the soil under the coffee
canopy where cherries infected with CBB would fall. Leucaena leucocephala hybrid KX2 or KX4 is a fast
growing nitrogen-fixing tree locally available to farmers, and its chipped mulch is an ideal mulching materials for coffee production. Two field trials were conducted to evaluate if Leucaena
mulch can enhance parasitism of indigenous EPNs on CBB and improve coffee production.
The results obtained thus far indicated that mulching of Leucaena leucocephala KX2/KX4 mulch increased infection of CBB recovered in the cherries on the ground by indigenous EPN by 2.82 fold in Trial I (at Poamoho Experiment Station) and 1.48 fold in Trial II (at Dole Plantation). Inundative release of lab reared indigenous EPNs into the soil (at 31,000 EPNs/ft2) either in mulched or unmulched plots did not further increase EPN infectivities on CBB. Although overall infection rate of EPN on CBB was only 22.9% and 9.76% in Poamoho and Dole trials, respectively, none-the-less, it would add to the current management options against CBB while helping the farmers to reduce fertilizer inputs, improving soil health condition through mulching, and likely lead to yield and profitability increases. We are progressing towards gathering coffee yield data with Dole plantation in a larger treatment scale to evaluate yield impact of KX2 mulching.
Preliminary pot trials were conducted to examine the “CBB bombs” technique with the help of a high school student. However, recovery of introduced EPN into CBB adults was next to zero infection. Thus, we modified the objectives to:
- Identify indigenous EPN species recovered from coffee orchards at Poamoho Research Station, and Dole’s Waialua Estate Coffee in Haleiwa, on the island of Oahu, Hawaii.
- Comparing virulence of indigenous EPN strains to Steinernema feltiae and Oscheius tipulae on CBB.
- Evaluate efficacy of N-fixing Leucaena mulch on enhancing infectivity of indigenous EPNs on CBB using both augmentative and conservative biological control methods.
- Examine the effects of Leucaena mulch treatments on plant and soil health.
Objective 1: Identify indigenous EPN species recovered from coffee orchards at Poamoho Research Station, and Dole’s Waialua Estate Coffee in Haleiwa, on the island of Oahu, Hawaii.
Soil samples were taken from coffee fields at Poamoho Experiment Station in Waialua, and Dole Plantation’s Waialua Estates Coffee fields in Haleiwa, Oahu, baited with meal worms. Infected meal worms with entomopathogenic nematodes (EPNs) recovered from the White Traps were isolated and maintain in water. Isolates were identified using Polymerase Chain Reaction (PCR) with universal primers for ITS regions of the nematodes. Nematode DNA sequences of each isolate were subjected to an NCBI Blast.
Objective 2: Comparing virulence of indigenous EPN strains to Steinernema feltiae on CBB.
The indigenous species (EPNs) Heterorhabditis H1, Heterorhabditis indica, and Oscheius tipulae isolated from our experimental sites or elsewhere from Hawaii were compared to the non-indigenous EPNs, Steinernema feltiae. In general CBB larval stage was more susceptible to EPN infection than the adult stage. Among which, Steinernema feltiae had the highest infection rate on CBB larva (15%). Only Heterorhabditis H1 species infected the CBB adult (5%). No other EPNs tested infected EPNs adults.
Objective 3: Evaluate efficacy of N-fixing Leucaena mulch on enhancing infectivity of indigenous EPNs against CBB using augmentative and conservative biological control methods.
A 2×2 factorial designed experiment was conducted at Poamoho Experiment Station in the Spring of 2018. The soil surface (under canopy) of coffee trees were 1) mulched (M) or not mulched (NM) with N-fixing tree mulch, Leucaena lucocephala KX2, and 2) drenched or not drenched with Heterorhabditis H1 (at 31,000 EPN/ft2). Each treatment had 3 replications. Heterorhabditis H1 inoculum was obtained from Poamoho soil, reared on meal worms in the lab, and drenched into the treatment plot under the coffee tree canopy with 1 Liter of water using a watering can. EPN infection rates on CBB over time was monitored using field cages made of 80-mesh strainer (Kleen-Rite Corp, Columbia, PA) where 5 coffee cherries naturally infested with CBB were enclosed in the field cage, buried into each treatment plots under the coffee canopy for one week. Samples were taken biweekly over 3 months.
Experiments were repeated but only with M vs NM treatments once at Poamoho (Fall 2017), and another one at Dole Plantation (Fall 2018). Experiment at Dole Plantation only lasted for 2 months but had 6 replications.
Objective 4: Examine the effects of Leucaena mulch treatments on plant and soil health.
During the Dole Plantation field trial, stem diameter and chlorophyll content of three branches per tree were monitored for plant health affected by mulching. Soil samples were also taken before initiation of mulching and at 2 months after mulching to determine the effects of mulching soil health. Total of 6 trees per treatments were monitored.
From Objective 1, we determined that the 7 EPN isolates recovered from Poamoho were identified to match Heterorhabditis sp. SGgi, Heterorhabditis sp. H1, and Oscheius sp. (tipulae), whereas the 4 EPN isolates recovered from Dole were all matching Oscheius sp. (tipulae). It is interesting to confirm in Objective 2 that CBB larval stage was more susceptible to EPN infection than the adult stage. Since CBB larval stage only present inside the coffee cherries, EPN biocontrol agents need to be delivered into the cherries to control the CBB effectively. However, only the adult CBB stage is moving in and out of the cherries to find new cherries to lay eggs. None-the-less, some EPNs manage to infect the adult stage which explained recovery of EPN infection on CBB larvae or adults inside the cherries in the field cages of Objective 3.
While it is encouraging to see that mulch had greater % of CBB with EPN recovery than no mulch (P ≤ 0.05), the inundative (augmentative) approach of introducing indigenous EPNs by drenching more lab reared EPNs into the soil had no effect in improving EPN infection. Soil moisture seems to play a role in enhancing EPN infection by mulching. We also observed more fungal infection on CBB in no mulch samples than mulched treatment. However, these fungi were not being identified. It is unclear if these are saprophytic fungi or entomopathogenic fungi.
When data was compared between Poamoho vs Dole trials, mortality of CBB in Leuceana mulch was greater at Poamoho than at Dole. This could be due to the present of Heterorhabditis sp. at Poamoho but not at Dole. Objective 2 suggested that the Heterorhabditis strains are more virulent than the Oscheius sp. (tipulae).
In Objective 4, Leuceana mulch increased stem diameters by 1.13% (P ≤ 0.05) and showed a trend in increasing canopy coverage by 2% monitored by %Cover© app. Though minimal, this improvement was observed in short period of time (2 months after mulching). We will ask the farmer to let us continue to monitor the effect over a longer term.
Based on nematode community analysis as soil health indicators, mulch improved soil food web structure with a significant increase in abundance of bacterivores, fungivores, and omnivores. Predatory nematode abundance was also increased though not significantly. What was most impressive was the dramatic increase of EPNs (P ≤ 0.01) from 13 to 780 EPNs/250 cm3 soil. This result supported that mulching with KX2 or KX4 can improve soil health and indigenous EPN abundance in short period of time. Future research need to future improve EPN infectivity on CBB.
Educational & Outreach Activities
A poster was created and presented at the Society of Nematologists 57th annual meeting in Albuquerque, NM on July 21-25, where nematologists and related professionals attended for an educational conference. My poster title was “Conservation biological control of coffee berry borer in Hawai`i by applying nitrogen fixing tree mulch to enhance efficacy of entomopathogenic nematodes.” I presented the details of my project and the results of my project to date.
A “Brown Bag” talk is currently being scheduled with the National Tropical Botanical Garden on the Island of Kaua’i for the end of January or early February, 2019. This will be a slide presentation for about 30-40 professionals that are either employees of the garden or the general public including Kaua`i Coffee’s, Jon Ching, one of my involved producers.
A paper will be finished by the end of February, 2019, and published in the College of Tropical Agriculture and Human Resources (CTAHR) Extension Publication (http://www.ctahr.hawaii.edu/site/Info.aspx). “Effects of Leucaena-KX2 tree mulch and the infectivity of entomopathogenic nematodes on coffee crop production.”
We hope to provide more options of sustainable agricultural practices for coffee production in Hawaii. Current intensive cherries and organic residues scrapping from the surface of the ground to ensure removal of infested coffee cherries from last harvest has a negative impact on soil health. Top soil organic matter was removed, and coffee farmers barely survived the labor intensive sanitation practices (tree stumping, branch clear stripping, and frequent biopesticide application) through government subsidies. Using N-fixing tree mulch as surface organic mulch would reduce the need for synthetic fertilizers that might leach into streams and oceans. The key outcomes of this project would be 1) mitigating CBB infestations in coffee farms; 2) a protocol for using Leucaena KX2 or KX4 as hedge rows, timing for pollarding trees and adding mulch to the fields; 3) improving soil health of coffee farms, 4) reduce soil erosion, and 6) new pest management strategies with applicability to CBB management elsewhere in the world.
Although indigenous EPN can be reared in the laboratory and be introduced into field soil, our field trial at Poamoho Experiment Station indicated that inundative release of EPN by soil drenching did not contribute to increase of EPN infection on CBB in the cherries on the ground. We had anticipated these results during the proposal development and had proposed to release EPN through lab infected CBB adult (“CBB bomb” technique where the CBB adults infected with EPNs will be released as biocontrol agent to carry EPNs into coffee cherries on the ground). However, several preliminary attempts to infect CBB adults in the laboratory resulted in no infection of CBB. Our second attempt was to release meal worm cadavers infected with indigenous EPN in the soil. We are in the process to analyze the data and will report this result in the final report.
It is encouraging that conservation biological control work in the field by introducing KX2/KX4 mulch. However, we learned that virulence of different species of indigenous EPN could affect the biological control results against CBB. The indigenous EPNs we isolated from the Poamoho Trial included Heterorhabditis sp. SGgi, Heterorhabditis sp. H1, and Oscheius tipulae, whereas that from Dole only contained O. tipulae. Oscheius was a weaker EPN than Heterorhabditis in terms of virulence on CBB as revealed in our lab trial. This explained why infection rates of EPN on CBB was higher in the Poamoho Trial than the Dole Plantation Trial. Based on our lab virulence assay trial, we hope to find another coffee farm with indigenous Steinernema feltiae as it had the highest infection rate among the species and strains of EPN tested against CBB.
Mulching the under canopy of coffee tree with KX2/KX4 mulch also significantly increased coffee growth (measured by stem diameter). We documented that KX4 mulch can improved soil food web structure within a short period of time (8 weeks) as indicated by the increase in abundance of bacterivores, fungivores, omnivores (significantly, P ≤ 0.05), and predatory nematodes though not significantly. These was revealed in a dramatic increase of EPNs (P ≥ 0.01) abundance in the soil from 13 in 250 cm3 of soil initially to 780 EPNs/250 cm3 soil at 8 weeks after mulching.
I would recommend using a N-fixing mulch like Leucaena KX2 or KX4 to reduce expensive sanitation practices, and unsustainable expensive biopesticide inputs. Mulching would improve plant and soil health and increase the number and infectivities of entomopathogenic nematodes in the soil as a natural enemies of coffee berry borers.