© 2003 Plant Management Network.
Gray Kernel Disease of Macadamia Nut:
Wendy S. Kaneshiro, Department of Plant and Environmental Protection Sciences; Catherine G. Cavaletto, Department of Tropical Plant and Soil Sciences; C. S. Tang, Department of Molecular Biosciences and Bioengineering; and Anne M. Alvarez, Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI 96822
Corresponding author: Anne M. Alvarez. firstname.lastname@example.org
Kaneshiro, W. S., Cavaletto, C. G., Tang, C. S., and Alvarez, A. M. 2003. Gray kernel disease of macadamia nut: Are bacteria involved? Online. Plant Health Progress doi:10.1094/PHP-2003-0825-01-HN.
Gray kernel disease of macadamia nut (Macadamia integrifolia Maiden and Betche), commonly called “bacterial nut,” occurs at levels of 0.7 to 10% of the harvested crop in commercial orchards in Hawaii during periods of high rainfall. Outwardly, diseased nuts appear healthy, but once opened, the kernels emit an acrid odor, possess a cheesy, acidic flavor, and are often uniformly gray (Fig. 1). While gray kernels are easily detected during the culling process, kernels expressing only a foul odor reach holding bins, where the odor is absorbed by healthy kernels, rendering the entire batch unmarketable.
Several bacterial species were previously associated with diseased kernels, but Koch’s postulates were not satisfied. To establish whether microorganisms are involved in disease development, 102 isolations were made from healthy and gray and/or foul-smelling kernels. Thin 1-cm sections were taken from the center of each kernel, soaked in sterile distilled water, and dilution streaked onto MS (1), SL (4), and modified TZC media (3). Bacteria were consistently isolated from both gray and foul-smelling kernels using MS and the modified TZC media, whereas fungi were rarely found. No bacteria were recovered from SL medium, which is selective for lactobacilli. Twenty-nine bacterial strains were isolated from symptomatic kernels, whereas only three strains were isolated from healthy kernels. The latter were recovered in very low numbers and were not associated with symptomatic kernels. The most frequently isolated strains were identified as Enterobacter cloacae (Jordan) Hormaeche and Edwards by API strips (BioMerieux Vitek, Inc., Hazelwood, MO) and/or the MicroLog™ identification system (release 4.2; Biolog, Inc., Hayward, CA). E. cloacae was not isolated from healthy kernels. Interestingly, E. cloacae is a pathogen of papaya (2) and ginger (Nishijima and Hepperly, pers. comm.) in the same region that gray kernel disease is problematic. E. cloacae decomposes sulfur-containing compounds generating hydrogen sulfide (H2S) from papaya fruits (5). The characteristic acrid odor from symptomatic macadamia nut kernels suggested breakdown of sulfur-containing compounds with a release of H2S and a deposit of iron sulfide (black) that discolored the kernel. Thirteen of the 29 strains isolated from symptomatic kernels, including all E. cloacae strains, produced H2S from ground macadamia kernels as detected by lead acetate strips.
Seven separate inoculation experiments were conducted on in-husk nuts, in-shell kernels and shelled kernels in an effort to satisfy Koch’s postulates and to determine effects of moisture, incubation time, and shell integrity on disease development. All bacterial strains recovered from symptomatic kernels were used in most experiments. Individual shelled kernels were stab-inoculated or soaked in a bacterial suspension (ca. 108 colony-forming units per ml). Prior to inoculation, in-husk nuts and in-shell kernels were surface-sterilized with 75% ethanol or 0.5% sodium hypochlorite. Depending on the experiment, one to five in-husk nuts or in-shell kernels were immersed in bacterial suspensions for 10 to 115 days. After incubation, shells were cracked and kernels were observed for symptoms.
All inoculated in-husk nuts remained symptomless. Gray kernels were reproduced twice on in-shell kernels with each of two E. cloacae strains. Strains identified as Phyllobacterium sp. and Sphingomonas sp. occasionally produced gray kernels, but were not recovered following inoculation. Reisolations from symptomatic kernels yielded predominantly E. cloacae even when they had been inoculated with other bacterial strains, suggesting internal contamination of the nuts prior to incubation. These data point to but do not prove a bacterial etiology for gray kernel disease, and implicate E. cloacae as one of the causal agents. Other bacteria also may be involved and possibly trigger a metabolic event that results in a uniformly gray kernel (Fig. 1).
Openings in the shell (naturally open micropyles or hairline cracks formed during sun exposure) were necessary for symptom development (Fig. 2). Failure of shell openings to reach the kernel may explain why some in-shell kernels did not develop symptoms following inoculation with E. cloacae. Kernels in intact shells remained symptomless even after immersion in bacterial suspensions for 115 days.
Moisture was required for symptom development, supporting field observations. Without added water, stab-inoculated shelled kernels did not turn gray up to 60 days after inoculation.
Exposure to the putative causal agent(s) probably occurs while nuts are on the ground awaiting harvest, in which case, strict adherence to the recommended four-week harvest interval (or possibly more frequent harvests) may reduce disease incidence by avoiding prolonged exposure of the nuts to moisture and soilborne bacteria. Meanwhile, further studies on disease etiology are warranted.
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