Host-based molecular markers for early detection of infection

  • Goal– To determine if host-based molecular markers are unique to a particular pathogen
  • Activities – Identification of which pathogens are present in grapevine wood

Host marker 1Host marker 2Water stress4 Results and Outputs – Utilize qPCR to detect differential expression of target genes Water stress 6Significant Outcomes & Impacts – Identification of which pathogens are present in nursery stock grapevine wood. Eliminate contaminated stocks that would otherwise be used for propagation.

Water stress and infection

Goal– To determine if water stress exacerbates infection and test specificity of host-based markers to infection

Activities – Identification of host-based markers specific to infection and water stress

Water stress2water stress 1 Water stress3Two watering regimes tested:

  1. Well-watered control
  2. Severe water stress (16-17 bars leaf water potential)

Three inoculation regimes tested:

  1. NINW: non-inoculated non-wounded
  2. NIW: non-inoculated wounded
  3. IW: inoculated wounded

 

Water stress4

Results & Outputs – To understand how abiotic stress can influence disease progression and to validate molecular marker specificity to Neofusicoccumparvum.

Water stress 5Water stress 6

Identification of new anatomical and biological resistance markers

Goal – Identify new anatomical and biochemical markers of resistance in grapevine wood.

Activities – Characterize changes in the wood that correspond to resistance to Eutypa dieback, using cultivars with known resistance/tolerance: Merlot (resistant), Cabernet Sauvignon (intermediate), and Thompson seedless (susceptible).

3.2

Results & Outputs – Identified differences in cell wall and xylem characteristics. Susceptible tablegrape Thompson seedless had low lignin and high glucan in its cell walls, whereas resistant winegrape Merlot had high lignin and low glucan (Cabernet Sauvignon was intermediate). Xylem vessel diameter also differed between these two cultivars; Thompson seedless had the largest vessels and Merlot had the smallest.

Significant Outcomes & Impacts – These markers of Eutypa resistance will compliment the detached-cane screening method, to provide additional measures of resistance. This is important because we are evaluating genetically-diverse germplasm, which may exhibit different modes of resistance. These markers are now being evaluated for the other trunk diseases (Botryosphaeria dieback, Esca, Phomopsis dieback), along with the detached-cane method of screening.

Accurate identification of trunk fungi

Goal 2 – Accurate identification of trunk fungi

Activities – Allow for proper disease management and control practices

1.5

Results & Outputs – Developed a comprehensive DNA sequence database of trunk inhabiting fungi

1.6 1.7

Significant Outcomes & Impacts – Accurate species identification will lead to proper disease management practices.

Pistachio Cultivar Selection

Goal – Identify new commercial cultivars with resistance to the most aggressive causal agents of Panicle blight of pistachio.

Activities
Evaluated resistance in all susceptible tissues (green tissues: panicles & fruit, woody tissues: stem) with the three most aggressive pathogens (Botryosphaeria dothidea, Neofusicoccum mediterraneum, and Lasiodiplodia citricola), either alone or in combination.  Inoculations were done on mature trees in an experimental orchard.

3.7

3.8

3.9

Results & Outputs
To identify resistant germplasm, stem inoculations are required.  Inoculation of green tissues leads to blighted panicles and fruit. However, results were not different among cultivars and all three pathogen species colonized equally. In contrast, inoculation of stems lead to cankers of different lengths, depending on the cultivar and the pathogen.

Kerman, which makes up >90% of California pistachio acreage, showed an intermediate level of resistance, with cankers ranging from 5 to 20 cm. Even more resistant (i.e., with smaller cankers) were new cultivars Golden Hills, Lost Hills, and Randy. New cultivars Kalehghouchi, Joley, and Aria were most susceptible than Kerman. Among the pathogens, Lasiodiplodia citricola was the most aggressive species (i.e., caused the largest cankers) on all seven cultivars, including Kerman.

Significant Outcomes & Impacts
Several new cultivars – Golden Hills, Lost Hills – are more resistant than Kerman. The inoculation method we developed will be important for evaluating preventative fungicides, esp. given that even the most resistant cultivars still develop cankers.

Infection in Almonds

Goals– To identify rates of wood-canker infections within almond orchards, determine management practices, identify the pathogens present in the orchard, and climate conditions conducive for sporulation in order to increase orchard 

Activities
Identification and surveying of infested orchards, isolation and identification of wood canker fungi, and spore trapping.

3.5s

3.6

Results & Outputs
Identified pruning wounds, growth cracks, and wood breakage serve as primary points of infection. Found that spore trapping of wood canker fungi is in association with rain events. Successfully extended these initial findings to almond growing clientele in CA.

Significant Outcomes & Impacts
Development of a preliminary model and management practices from field observations and spore trappings.

Almond Resistant Cultivar Selection

Goal – Identify almond cultivars with resistance to the most aggressive causal agents of wood-canker diseases of almond: Band canker and Lower limb dieback.

Activities – Compared virulence of two Neofusicoccum species inoculated to the woody stems of potted almond plants.

3.3

3.4

Results & Outputs
Peerless was the most resistant to both species of the pathogen. Carmel, Sonora, and Padre were among most susceptible, with some differences depending on the species. Susceptible cultivars had the highest mortality. Cankers were so severe in Sonora that 50% of the trees died after 10 months. This means that infections of young trees, esp. of susceptible cultivars, must be protected in young orchards.

Significant Outcomes & Impacts
Now that we know the causal agents, we must identify preventative practices.

Development of Screening Technique

Goal – Develop a rapid and reliable screening technique for resistant grape germplasm.

Activities – Compared screening methods for resistance to Eutypa dieback among nine genetically-diverse cultivars.

3.1

Results & Outputs
Merlot and Primitivo were most resistant to Eutypa dieback, based on both methods (hardwood cuttings and detached canes). However, the detached-cane method – a rapid alternative that takes only 5 weeks – differentiated cultivars similarly to the hardwood-cutting method, which is the standard in most labs.

The susceptible control, table-grape cultivar Thompson seedless, was ‘beat’ by an even more susceptible table grape cultivar, Husseine. Fortunately, this is not a commercial table grape, but it can serve as a new susceptible control in our studies.

Significant Outcomes & Impacts
The detached-cane method will serve as a phenotyping assay for use with materials from VitisGen and other grape breeding projects. In this way, our SCRI project builds on other SCRI projects that have already completed major genotyping efforts.

Mobile diagnostic aid application development

Goal – Develop visual diagnostic aids for trunk diseases.

Activities—Create a web/mobile application and website for trunk disease diagnosis.

2.5
Results & Outputs
Developed beta-versions of logic and flow charts.

2.6

Significant Outcomes & Impacts
Accurate disease diagnosis will minimize losses by leading growers to proper management practices.

Link: Trunk Disease Management for Eastern grape growing regions

Identify the socioeconomic hurdles to adoption

Goal – Increase early adoption of preventative practices.

Activities – Surveyed growers on usage of three practices and developed an economic model to predict cumulative returns (ie profits) when adopted early (year 3) vs. late (year 10).

2.1 2.2 2.3

Results and Outputs – Despite low efficacy of late adoption, >50% of growers adopt practices after year 8. Why? Profits are low in year 3, and the cost of preventative practices seems relatively high, esp. because trunk diseases are not yet serious. Regardless, early adoption (not late adoption) increases profits in the long term.2.4

Significant Outcomes & Impacts – Reducing uncertainty about economic benefits in the long term will increase adoption of preventative practices in young vineyards.

For more information from Vicken’s research group, please visit: Research Briefs on regional grower surveys and economic analyses

Links: