Phosphorous Acid Injections and Soil Drench Applications Can Control Macadamia Quick Decline

LISA M. KEITH1*, LIONEL S. SUGIYAMA1, TRACIE K. MATSUMOTO1 AND MIKE A. NAGAO2

1 Tropical Plant Genetic Resource and Disease Research Unit, Daniel K. Inouye United States Pacific Basin Agricultural Research Center, USDA-ARS, Hilo, Hawaii. 2 Researcher Emeritus, Komohana Research Extension Center, University of Hawaii at Manoa, Hilo, Hawaii.

Abstract: Macadamia quick decline (MQD) threatens commercial and residential production of macadamia (Macadamia integrifolia) in Hawaii. To identify strategies to manage the disease, fungicide and application measures were evaluated. Fosphite® was examined in vitro and inhibited radial growth of Phytophthora tropicalis on fungicide- amended 10% V8 juice agar; the concentration at which growth was reduced to 10% was ≥1.3 mL/L. In field studies, a soil drench application of Fosphite® was compared with passive and active injection methods. Results indicate that drenches and/or tree injections with phosphorous acid fungicides can arrest the progression of the MQD symptoms in infected trees, eliminate visible symptoms and prolong their productive life, in some instances for as much as 1000 additional days.

Key words: Macadamia, Phytophthora tropicalis, Fosphite®.

Introduction et al., 1991). MQD has been a persistent problem since 1986 when it started killing productive 14- to Macadamia (Macadamia integrifolia Maiden & Betche) 36-year-old macadamia trees in the commercial- are evergreen trees that can grow to heights of 19 m growing region surrounding Hilo, Hawaii (Oi et al., with canopy widths of 13 m (Nagao, 2011). M. 1991). Fungi including Nectria regulosa, Xylaria integrifolia leaves, which occur in whorls of three, are arbuscula, Phellinus gilvus, and Acremonium recifei have light green or bronze colored when young and are been attributed to MQD and can kill twigs on 10–15 cm in length with about a 1.3 cm long petiole healthy macadamia trees after artificial inoculation when mature (Nagao, 2011). Macadamia nuts are (Ko, 2009). Keith et al. (2010) were the first to renowned as being among the world’s most report Phytophthora tropicalis as the primary causal delicious. Whole kernels are sold as oil- or dry- agent of MQD and its association with sap bleeding, roasted snack nuts, while broken kernels are used as ambrosia beetles, and a saprotrophic species of an ingredient in a variety of products including ice Nectria. P. tropicalis also causes raceme and foliar cream, cookies and bread (Ko, 2009). Macadamia blight in macadamia and may serve as a primary nut production in Hawaii is a $38.2 million source of inoculum for MQD by surviving on twigs industry producing an estimated 49 million or stem cankers (Ko, 2009). pounds net of wet-in-shell nuts (NASS, 2012). Prior to 1986, the macadamia nut industry in In the 1990s, MQD caused loss of approximately Hawaii had not encountered disease problems 25,000 trees in a single orchard over the course of a that posed a serious economic threat; however, in five-year period (M. Nagao, personal 1986 many dead and rapidly declining trees were communication). MQD continues to be a serious observed in orchards located on the east side of problem in Hawaii. Because MQD primarily affects Hawaii Island (Oi et al., 1991). The disease, older trees, minimizing tree loss in mature orchards which was first designated as “macadamia quick is critical for maintaining the economic viability of decline” (MQD) by Hirae (1989), was new to Hawaii’s macadamia industry. The objectives of this macadamia in Hawaii and had not been reported in study were to: (i) determine in vitro efficacy of other countries growing macadamia (Oi *Corresponding author, Email: [email protected]

1 Pac. Agric. Nat. Resour. 2014 Vol. 4: 1-7 2 Phosphorous Acid Injections and Soil Drench Applications Can Control Macadamia Quick Decline fungicides currently registered for use 37.84 mL/L of water. The solution was mixed in an on macadamia in Hawaii to control P. 18.9 L bucket and poured uniformly in a 3.0 m tropicalis; and (ii) compare efficacy of different field diameter around the base of the tree trunk. Seven application methods for control of MQD. trees were treated with four drench applications. The first two applications were applied a month apart; the third application was 3 months later Materials and Methods followed by the fourth a month later.

Field observations and symptoms Trunk passive injection A field plot planted in 1991 with the macadamia nut Four holes spaced equidistant to each other around variety HAES 333 located at the University of the circumference of the tree were drilled in a Hawaii Waiakea Experiment Station was observed downward angle (approximately 60° towards the for signs (trunk bleeding, frass and/or Nectria sp.) center of the tree) to a depth of 38.1 mm at chest and symptoms (yellowing or browning of leaves in height using a cordless drill with a standard 11/32” the canopy) of MQD. In May 2006, seven trees (8.7 mm) drill bit. The Fosphite® fungicide (label suspected of MQD were tagged and observed rate for trunk injection is 15 mL of undiluted weekly. At the conclusion of the trial in January product for every 50.8 mm of trunk diameter at 2008, 34 trees had been tagged. When the field was breast height (DBH), approximately 1.4 m from the revisited in August 2010, 44 trees suspected of or canopy floor) was applied slowly and equally to each killed by MQD were identified. Upon identifying a hole using a 50 ml syringe with a blunt tip 16 gauge tree with MQD the vertical infection zone (high and needle. low point on the trunk with visible signs) was marked at the two ends with a galvanized nail and a Trunk active injection small piece of flagging tape. The markings provided This method utilized compressed air and a leak a consistent starting point to measure any expansion proof proprietary plug to forcibly inject trees with of the infection zone. Disease progression was the fungicide (label rate equal to passive injection; measured in linear mm from these points. Arborjet® Tree I.V. System, Woburn, MA). Four equidistant, 7/32” (5.6 mm) diameter holes were In vitro fungicide efficacy tests drilled 5/8” (15.9 mm) below the cambial layer and Radial growth tests were conducted with six isolates 304.8 mm above the trunk flare. Arborjet® plugs of P. tropicalis (LS06-1 to LS06-6) to determine the were inserted, a blunt syringe was attached and a efficacy of Fosphite® (mono- and dipotassium salts pressurized container containing concentrated of phosphorous acid, 53%; JH Biotech, Inc., Fosphite® was inserted into the plug. The valves Ventura, CA) at 0, 0.2, 0.5, 1.0, 1.9 and 3.8 L/378.5 were slowly opened to release the solution to each L. Inoculum was grown on 10% V8 juice agar for 7 plug uniformly. days at 24°C under continuous fluorescent lighting. An inoculum plug was excised using a #4 cork borer Dual treatment and placed at one edge of a large petri dish Three trees received a two drench treatment that containing 10% V8 juice agar amended with was applied a month apart followed by a passive or Fosphite®. At days 4 and 7 radial growth active injection at the rates described above as the measurements were taken using a Storm digital symptoms became more severe. caliper (Central Tools, Cranston, RI). Two radial growth measurements were taken from the edge of Field Studies the inoculum plug to the end of the advancing margin. Three petri plates per isolate and treatment Field treatments were repeated 6 times over 2 were used. The test was conducted twice. seasons, which corresponded to the occurrence of new, visible signs and symptoms of MQD. Results Soil drench indicate the average of each treatment over the entire length of the study. To simulate a drench application Fosphite® was applied at label rate of 4 quarts per 100 gallons or

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Fig. 1. Symptoms and signs of macadamia quick decline. A. Trees exhibiting dull green, yellow and brown leaves within the tree canopy. B. Orange fruiting bodies of Nectria. C. The presence of ambrosia beetle frass. D. Trunk bleeding.

of 10.1 ± 2.8 and 4.4 ± 2.3 mm occurred at the 0.2 Results and 0.5 L/378.5 L rates, respectively. No growth was observed at the 1.0 L/378.5 L rate as well as the Field observations and symptoms drench label rates of 1.9 and 3.8 L/378.5 L. Trees exhibiting dull green, yellow, or brown leaves within the tree canopy were observed. Trees Field treatments affected by MQD exhibited excessive sap bleeding Field treatments are summarized in Table 1. Using from the trunk, frass from ambrosia beetle feeding, the passive injection method, approximately 24 to and orange fruiting bodies of the fungus Nectria 32% of the calculated volume was absorbed per rugulosa; yellowing and browning of the leaves within tree, taking approximately 30 minutes to an hour. the tree canopy soon followed (Fig. 1). At the start With the Arborjet® system, 100% of the calculated of the experiment in May 2006, the plot contained a volume was injected into each tree within 5 minutes. total of 97 trees with 7 trees (7.2%) suspected of having MQD. At the end of the weekly observations (19 months), the number of trees affected by MQD Table 1. Treatment summary was 34 trees (35.1%); 13 trees (13.4%) were killed # Fosphite® # Trees by MQD. Treatment applications Treated Soil drench 4 7 In vitro fungicide efficacy tests Passive injection 1 3 At day four after inoculation, radial growth was Active injection 1 2 observed at 0, 0.2 and 0.5 L/378.5 L rates. The Active injection 2 2 average radial growth ± the standard error of the Dual Treatment: means (SEM) of the P. tropicalis isolates were 67.6 ± Drench + Passive 2 + 1 1 2.0, 6.5 ± 2.2 and 2.3 ± 1.8 mm, respectively. No injection 2 + 1 2 growth occurred at 1.0, 1.9 or 3.8 L/378.5 L. At 7 Drench + Active days, complete plate growth of 83.0 ± 0.0 mm injection occurred on the control plates. Mean radial growth

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control/untreated trees died resulting in a 100% mortality rate. Tree death occurred within an Figure 2 shows the progression of MQD in the field average of 202.5 ± 18.3 days with a range of 149 to over the length of the experiment. All six of the 254 from the first signs of MQD to death. died within 204 days from the first signs of MQD. The surviving trees (which received two to four drench applications) were observed for an average of 580 days. It took approximately 448 days for all symptoms to clear. The trees remained symptom- free for an average of 170 days. The two trees treated with only two active injection applications survived for 259 days, the duration of the test, but had no symptom-free days. The three trees treated with a single passive injection were observed for 294 days. The surviving trees remained symptom free for only an additional 26 and 85 days. Trees receiving Fosphite® injections survived 3.6 times longer than the control trees. Three trees treated with two drench applications and a single trunk injection (active or passive) survived 4 times longer than the untreated trees. The tree treated with two drench applications and one active injection had no symptom-free days, while the tree treated with two drench applications and a single passive injection remained symptom-free for 49 days. Table 2 summarizes the field data and shows the overall effectiveness of the treatments. Fig. 2. MQD field progression over time. Survey of Waiakea ‘333’ field. Numbers indicate the row and tree Disease progression number. Date of survey: 5/24/06 (A), 5/31/07 (B), Results indicate that injection methods (215.9 ± 1/4/08 (C), 8/13/10 (D). Grey square = gap, no tree present; yellow square = possible MQD tree; red square 78.7 mm) and drench applications (332.7 ± 132.1 = dead tree; green square = new MQD. mm) were effective at slowing down symptom development. As expected, the control (no The trees treated by more than one drench survived treatment) resulted in the widest spread of at least 5 times longer before dying when compared symptoms (2415.4 ± 322.58 mm of disease to the length of the survival time for control trees. progression) over the length of the study. The dead tree (receiving a single drench application)

Table 2. Effect of Fosphite® drench and injections on survival of trees with MQD

Method of Treatment Drench & None Injectc Drench Totald Inject Number of trees receiving 6 7 7 3 17 treatment Number of trees surviving 0 2 6 1 9 Survival length (days)a 202.5 ± 18.3b 719.3 ± 98.7 1025.1 ± 136.7 817.3 ± 202.9 862.5 ± 80.9 a Length of survival from initial observation of MQD signs and/or symptoms to tree death b Mean ± SEM c Passive or active injection d Trees receiving Fosphite® treatment(s)

Pac. Agric. Nat. Resour. 2014 Vol. 4: 1-7 Lisa M. Keith et al. 5 Discussion fungicide resulted in similar levels of inhibition (Keith, unpublished). Since fungi were the suspected pathogen in MQD, efficacy studies were conducted in 1991 with the Trees that were forcibly injected had a slightly fungicides Ridomil® 2E (21.5% metalaxyl; longer survival rate as compared to the passively Syngenta crop protection, Greensboro, NC), injected treatment; this could be due to the amount Kocide® DF (61.4% copper hydroxide; Griffin of Fosphite® absorbed per tree. Macadamia trees L.L.C., Valdosta, GA), Aliette® (80% fosetyl-AI; with at least one forcibly injected treatment had no Rhone-Poulenc Ag. Co., Greensboro, NC) and symptom free days while trees injected with at least Champ® (23.0% copper hydroxide; Nufarm one passive application had an average of 53 days Americas Inc., Burr Ridge, IL) to determine their with no visible symptoms. With the passive effectiveness in controlling MQD. It was concluded injection application the tree that died only received that Champ® and Ridomil® 2E may be useful as a less than a quarter of the label rate while the trees preventative treatment (Nagao et al. 1992). that survived received a minimum of 28% of the Increased survival of the trees and a healthier label rate. For the surviving trees, trunk bleeding appearance associated with Aliette® and Ridomil® decreased first followed by a slow decline in either suggested that Phytophthora may be playing a role in the presence of frass and/or the orange perithecia the cause of MQD (Nagao et al., 1992). In 1995 it of Nectria. In instances when there was no control, was reported that excessive moisture and lack of trunk bleeding continued or increased; the number adequate drainage were critical factors in causing of powder posts from ambrosia beetles and Nectria MQD (Nishijima et al., 1995). In 1997, experiments perithecia also increased until death of the tree with water logged seedlings inoculated with P. capsici occurred. and treated with Ridomol® 2E and WECO-42894 (a systemic experimental fungicide) were examined. The Waiakea Experiment field trial site was revisited The study concluded that oxygen stress caused by 1575 days after the study was initiated. Two of the poor drainage, excessive rain and other factors was 7 trees that were drenched with Fosphite® the primary cause of MQD and P. capsici was continued to survive and remained symptom-free secondary in nature (Nishijima et al., 1997). It wasn’t (one tree that was drenched 4 times and one tree until the MQD issue was readdressed that the causal that was drenched twice and injected at 37% of label agent of the disease was determined and confirmed rate). One tree that received one passive injection at by Koch’s postulates (Keith et al., 2010). 28% also remained symptom free. In contrast, 5 of the 6 trees which were forcibly injected using the Use of phosphite fungicides has become common Arborjet® system either once or twice died and the practice in some agricultural or orchard crops (such remaining tree has all of the classical symptoms of as citrus, avocado, cacao and oak trees) severely MQD. On average it took about 222 days for all affected by Phytophthora diseases (Darvas et al., 1983; visible symptoms on the trunk to clear up once Garbelotto et al., 2007; Guest et al., 1994; Timmer, Fosphite® was applied; however, symptoms 2002; Wood et al., 1987a; Wood et al., 1987b). reappeared over time indicating that phosphorous Phosphite treatments may lead to increased healing acid applications for macadamia are not completely of diseased trees by slowing the growth of the curative, which agrees with the study by Norman et pathogen while simultaneously enhancing growth of al. (2006). When MQD symptoms reappeared the plant host and compartmentalizing lesions during the course of the study (i.e. at the first caused by Phytophthora (Garbelotto et al., 2007; indication of trunk bleeding), additional Fosphite® Guest and Grant, 1991). Fosphite®, the mono- and treatments were applied (2 drenches within six di-potassium salts of phosphorous acid, is labeled to months of each other) and symptoms subsequently control Phytophthora at 1.9-2.9 L/378.5 L and is disappeared. It often took an additional 116 to 338 registered for use on macadamia nut in Hawaii. total days for all symptoms to completely disappear Radial growth trials comparing the fungicide after Fosphite® application. Our results show that Fosphite® at 200 ppm (a.i.) incorporated into 10% treatments consisting of 2 to 4 drench applications V8 juice agar medium reduced the growth of P. within a 6 month period or an injection that can tropicalis by about 95% as compared to 10% V8 juice deliver at least 28% of the label rate are effective agar alone. Leaf bioassays using microcentrifuge treatments for controlling MQD. According to the tubes filled with 200 ppm (a.i.) of Fosphite® study by Hardy et al. (2001), phosphate treatments

Pac. Agric. Nat. Resour. 2014 Vol. 4: 1-7 6 Phosphorous Acid Injections and Soil Drench Applications Can Control Macadamia Quick Decline can be effective for 2 to 6 years; however, the length Darvas, J.M., Toerien, J.C. and Milne, D.L. 1983. Injection of efficacy of treatments was not that long for of established avocado trees for the effective control of macadamia and re-application was necessary. A Phytophthora root rot. California Avocado Society Yearbook 67: 141-146. study by Rizzo and Garbelotto (2003) showed that phosphate treatments were effective at controlling Garbelotto, M., Schmidt, D.J. and Harnik, T.Y. 2007. sudden oak death (SOD) for 8 months and they Phosphite injections and bark application of phosphite + pentrabark control sudden oak death in coast live recommended yearly fungicide treatments. oaks. Arboriculture and Urban Forestry 33:309-317. Guest, D. and Grant, B. 1991. The complex action of Conclusions phosphonates as antifungal agents. Biol. Rev. 66:159- Our research has provided vital information on the 187. etiology of MQD and its management. Prior to our Guest, D.I., Anderson, R.D., Foard, H.J., Phillips, D., study, when trees possessed MQD symptoms, the Worboys, S. and Middleton, R.M. 1994. Long-term only recommendation was to remove the tree to control of Phytophthora diseases of cocoa using trunk- reduce the inoculum level in the orchard. injected phosphonate. Plant Pathol. 43(3):479-492. Knowledge that Phytophthora infection of the trunk Hardy, G., Barrett, S. and Shearer, B.L. 2001. The future of was the cause of MQD has led to the development phosphite as a fungicide to control the soilborne of a control strategy that can target this pathogen pathogen Phytophthora cinnamomi in natural ecosystems. (Keith et al., 2010). We have demonstrated that Australasian Plant Pathol. 30:133–139. drenches and/or tree injections with phosphorous Hirae, H.H. 1989. MQD = Quick death. Hawaii Mac Facts acid fungicides can arrest progression of MQD 2(4):1. symptoms in infected trees and prolong their Keith, L., Sugiyama, L. and Nagao, M. 2010. Macadamia productive life. Early detection (trunk bleeding, quick decline caused by Phytophthora tropicalis is frass and/or Nectria) and treatment, along with associated with sap bleeding, frass, and Nectria in monthly field scouting, is critical. In our study, Hawaii. Plant Dis. 94:128. treated trees (drenching with or without injection) Ko, W.-H. 2009. Nature of slow and quick decline of lived an additional 700+ days which allowed for two macadamia trees. Bot. Stud. 50:1-10. additional harvests of approximately 45.5 kg of in- Nagao, M.A. 2011 (revised). Farm and Forestry Production shell nuts per tree per year, resulting in additional and Marketing Profile for Macadamia Nut (Macadamia income of approximately $100 per tree per year. integrifolia and M. tetraphylla). In: Elevitch, C.R. (ed.). There is currently 6879.7 ha (175 trees per ha) of Specialty Crops for Pacific Island Agroforestry. macadamia grown in the state of Hawaii Permanent Agriculture Resources (PAR), Holualoa, (NASS, 2012). The trunk injection/drench control Hawai‘i. http://agroforestry.net/scps method provides the macadamia industry a Nagao, M.A., Yoshimura, E.R., Nishijima, W., Hirae, H., solution to MQD and describes an application Santos, G.L. and Hara, A. 1992. MQD Update. Hawaii technique that could be used for other root or Macadamia Nut Association, 32nd Annual Conference trunk problems in macadamia and other fruit/ Proceedings, pg. 28. nut trees. Continued research to optimize National Agricultural Statistics Service (NASS), USDA, application strategies on a larger scale is Hawaii Field Office. 2012. Hawaii Macadamia Nuts, needed. Final Season Estimates, released July 13, 2012. NASS, USDA, and Hawaii Department of Agriculture, Acknowledgments Agricultural Development Div., Honolulu, Hawaii, We thank D. Aoki, B. Bushe and R. Kai for their http://www.nass.usda.gov/Statistics_by_State/Hawaii /Publications/Fruits_and_Nuts/mac-finFF.pdf excellent technical assistance and P. Follett and M. Wall for their pre-submission reviews. Nishijima, W., Yamaguchi, A., Sako, G. and Akiyama, M. 1995. Update on macadamia quick decline research. Hawaii Macadamia Nut Association, 35nd Annual Disclaimer Conference Proceedings, pg. 34. Mention of trademark, proprietary product, or vendor Nishijima, W., Yalemar, J. and Tamimi, Y. 1997. Macadamia does not constitute a guarantee or warranty of the quick decline research update. Hawaii Macadamia Nut product by the U.S. Dept. of Agriculture and does not Association, 37th Annual Conference Proceedings, pg. imply its approval to the exclusion of other products 21. or vendors that also may be suitable. Norman, D.J., Chen, J., Yuen, J.M.F., Mangravita-Novo, A., Byrne, D. and Walsh, L. 2006. Control of bacterial wilt References of geranium with phosphorous acid. Plant Dis. 90:798- 802.

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Oi, D., Ueunten, G., Yamaguchi, A., Nagao, M., Hara, A. Wood, R., Bennett, I.C. and Blanken, P.A. 1987a. Injectable and Nishijima, W. 1991. Quick tree decline: a new formulations of phosetyl-Al developed for root rot problem of macadamia in Hawaii. Hort. Sci. 26(5):560- control in avocado trees in South Africa. South African 561. Avocado Growers’ Association Yearbook. Proceedings of the First World Avocado Congress 10:97- 99. Rizzo, D.M. and Garbelotto, M. 2003. Sudden oak death: Endangering California and Oregon forest ecosystems. Wood, R., Bennett, I.C., Blanken, P.A. and Grech, N. 1987b. Front. Ecol. Environ. 1:197-204. Control of Phytophthora root rot in citrus trees by injecting phosetyl-Al into the stems. Proceedings of the Timmer, L.W., ed. 2002. Florida Citrus Pest Management Twenty-fifth Annual Congress of the South African Guide. Univ. Fla. Inst. Food Agric. Sci. Publ. No. SP- Society for Plant Pathology, 20 p. 43.

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