608 Viability Testing for Rapid ʽŌhiʽa Death Found On Ambrosia Beetles and Their Frass

Kelly Jaenecke, Kylle Roy, Bob Peck, Carter Atkinson

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Background

 

  • Ambrosia beetles (Curculionidae: Scolytinae) are hypothesized to play an important role in the spread of the fungal pathogens Ceratocystis lukuohia and C. huliohia that cause Rapid ʽŌhiʽa Death in Hawaiʽi.
  • We reared beetles in the laboratory from portions of freshly cut ‘ōhiʽa that were already infected with Ceratocystis and contained active beetle galleries.
  • We reared 7 ambrosia beetle species from infected ‘ōhiʽa trees on Hawaiʽi Island.
  • We employed a variety of culturing techniques to investigate the overall viability of Ceratocystis propagules on ambrosia beetles and their frass that emerged from ‘ōhiʽa.
  • We successfully cultured Ceratocystis from:
    • frass collected throughout the length of naturally infected ‘ōhiʽa trees.
    • ambrosia beetles collected from individual galleries on bolts in the lab, and from wild caught beetles on sticky cards in the field.
  • We are continuing to investigate the role of ambrosia beetles in the transmission of Ceratocystis focusing on beetles and frass collected from emergence traps in the field.
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Modes of Transmission

Indirect Transmission: The beetles may indirectly spread the fungus through propagules attached to macerated boring material (frass) that is released into the environment during their excavation of reproductive galleries. Frass produced by beetles that is expelled from ohia can then be picked up and transported elsewhere (neighboring trees and beyond).

 

 

Direct Transmission: The beetles may directly spread the disease between trees by transferring viable fungal propagules attached to their bodies.

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Carrot Bait Method
Standard carrot baiting is a well-used method for culturing Ceratocystis. This method is widely used to test viability in beetle frass, sawdust, infected wood, and other materials, but does not work well for low quantities of spores. 

 

A small amount (5-20 mg) of frass is collected.  Carrots are surface-cleaned with dH20 and 70% ethanol,  peeled, rinsed, and sliced into thin (0.5 cm) discs. Frass is put on one disc, and sterile water is sprayed on a second disc of equal size. The discs are then sandwiched and held together with a strip of parafilm. 

 

Carrot discs are inspected for growth  every week. Ceratocystis can be identified by presence of fruiting perithecial bodies that produce sticky spore masses. Perithecia (or fungal mats) can then be subcultured on V8 media to confirm identification.  

 

Once DNA is extracted from fungal growht, we can use a qPCR genetic test to provide confirmation of Ceratocystis presence and a determination of what species is present (C. lukuohia and/or C. huliohia). 

 

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Water Bath Method
One drawback of carrot baiting is that the sensitivity was not great enough to pick up small amounts of spores. The water bath is a modification of the carrot bait method. It uses more water and oxygen along with some antibiotics to encourage the survival of potentially very low numbers of spores. This test works well to test viability on individual beetles and microscope slides taken from aerial spore samplers. The tubes were fixed with glass pasteur pipettes with rubber stoppers to keep them suspended near the water surface. Tubes were organized with aquarium tube splitters and zip ties in order to accommodate more samples simultaneously.

 

Sticky cards trap beetles as they fly to a wounded ‘ōhiʽa. The beetles are  tested for presence of fungus using the water bath viability test.

 

Beetles were added to the water bath in groups of 1-14, depending on the emergence of the sample (same gallery, or same sticky card). Beetles were collected directly from galleries (boring dust traps on bolts in the lab) and  from sticky cards in the field.

 

Microscope slides collected from aerial spore samplers are also being tested for presence of viable Ceratocystis.

 

Presence of C. lukuohia and C. huliohia can be determined by perithecial growth on the carrots, and confirmed using qPCR reactions.
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Sawdust Media
We modified a V8 media that already works well to culture Ceratocystis in order to make it more enticing to live beetles. We added 33 g sterilized ohia sawdust, 10 g agar, 25 ml V8, 225 ml dH2O, and 2.4 ml ethanol as an attractant. 

 

We poured media into glass petri dishes (beetles can chew through polystyrene!). Moisture content was an important factor since the beetles can be trapped in tiny water droplets formed by condensation. To combat this, the media was dried in a low-temp (30C) drying oven for 3-5 days.

 

The media was effective at enticing beetles to bore in. Galleries were created on most occasions, and eggs were laid in a few instances. 

 

Fungal mats grew quickly on the surface of the media and inside galleries. No Ceratocystis was identified from any cultures. Competition from other fungus could not be ruled out as a limiting factor, and the method was deemed insufficient in determining the presence of viable Ceratocystis.
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Viability Testing Results
A small percentage of both lab reared and wild caught beetles were positive for viable Ceratocystis. Of 9  positive viability results, 8 were C. lukuohia and 1 was C. huliohia. This is one example of how data collected using the water bath method were used to identify the occurrence of ROD causing fungi on the bodies of beetles. 
Viable frass was produced from nearly all tree collected, and from a range of heights along the trees. This is one example of how the carrot bait method was used to produce a dataset on frass viability at different heights on a selection of trees on Hawaii Island.

 

The water bath method was also used to test for the presence of viable frass collected from aerial spore traps: 

  • Out of 156 slides, we found 413 woody frass particles
  • We observed fungal structures 3 times
  • No viable Ceratocystis was detected
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