Week 6: July 6 – 12

On Monday, Beth and I went out to the block of V. rupestris B38 x ‘Horizon’ to rate for disease. To our relief, disease there was!

BRcluster3 BRcluster1

Now that we have disease, we can phenotype the population, one of the most important steps in creating molecular markers. A phenotype(n.) is a trait of an individual, due to a mix of genetic and environmental factors. As we are looking at the inheritance of black rot resistance in grapevines, our phenotype of interest is disease severity on clusters. We therefore phenotype(v.) the population by rating disease on a 0 to 4 scale

0 – No sign of disease

1 – First sign of disease: a few lesions or starts of lesions on fruit

2 – Few to many lesions on fruit, with no pycnidia formed

3 – Few to many lesions on fruit, some lesions have pycnidia

4 – Tons of lesions with many pycnidia

Rating for disease resistance on the clusters is actually pretty challenging, because you have to check every single berry on the cluster to make sure it doesn’t have a lesion that could bump it up to the next category. It doesn’t sound too hard, but there’s something about the three-dimensionality of the clusters that makes it hard to tell. Also, as you can tell from the classifications, it is a bit subjective. And the ratings take forever. I am not very good at them, but Beth is quite good at it.

Besides the disease ratings, the week was not very productive, because Beth and I both got wiped out by a stomach bug.

On Wednesday I did a few secondary isolations off the second batch of plates I’d made last week. I do this by taking a chunk of of one of the many (different kinds of) colonies on the primary isolation plate, and moving it to a fresh sterile plate where it can be alone. We had better luck with the second batch than the first: a few more of the colonies looked like they could have been black rot. To define whether or not they actually are, we need to analyze the genetics, but that is an activity for a later day.

Editors note: I now only have one week left in the program, and we haven’t had time to do the analysis, so the world may never know. I may as well tell you now, dear reader, so you don’t get your hopes up.

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Week 5: June 29 – July 5

This week was a bit of a waiting game: we waited until later into the week for the fungi isolated last Friday to start to grow so we could evaluate what we collected. In the meantime, Beth and I collected a few more leaf/fungal samples, sterilized them, and did a few more isolations.

So on Wednesday I checked up on my plates, and what I saw made me wish I hadn’t. There was bacterial contamination, as well as contamination by other fungi on most plates, and a few colonies that looked like they could have been G. bidwellii if you squinted at them right, but probably weren’t.

Bacterial and fungal contamination on my isolation plates
Bacterial and fungal contamination on my isolation plates

Beth tells me that the initial isolations always look this bad, but I nonetheless felt a little disheartened. I hoped that the isolations we did the day before turned out a bit better.

We were also waiting this week for the inoculated V. rupestris B38 x ‘Horizon’ population to start to show symptoms of infection. Infection starts to become visible at 11 to 16 days, and having inoculated on June 16, we were expecting to see symptoms any day now.

We were disappointed. On Thursday July 2, now 16 days post inoculation, we scoured the block for symptoms and found only one diseased vine, probably the result of an earlier infection and the product of naturally-occurring inoculum. We started to speculate as to what could have gone wrong–could the inoculum not have taken to the plants, for one reason or another, or was it just delayed by the cooler-than-average weather? Only time would tell, and we just prayed to the science gods that when we checked again on Monday there would be disease.

This week was one of those unfulfilling weeks where nothing seems to go your way. If only G. bidwellii would grow this poorly in commercial vineyards, we could all go home early instead of sitting around scratching our heads 😉

To come: updates on what became of the fungi. Stay tuned!

Week 4: June 22 – 28

On Monday and Tuesday, I helped again with another leaf disk assay, though this time I worked not just with Konstantin, but with the VitisGen Powdery Mildew Phenotyping Center.

[IMAGE PENDING APPROVAL-to be updated]

VitisGen, which I’ve been alluding to in a few of my posts but have not actually addressed, is a collaborative network consisting of grape breeders, plant pathologists, USDA personnel, and faculty/staff from many universities, who research ways to streamline the grape breeding process, primarily by integrating genotypic and phenotypic data to develop molecular markers. Here at the station, I work with members from Cornell University and USDA-ARS. Molecular markers are DNA sequences within the plant’s genome that are associated with a trait. By identifying molecular markers for target traits, plant breeders can screen undesirable individuals more efficiently, saving time, money, and land.

The traits VitisGen focuses their efforts on are those of the most significance to growers and breeders. The 3 most important traits are low temperature responses, fruit quality, and powdery mildew resistance, though other diseases and traits are also investigated as part of a local phenotyping effort. My and Beth’s project is such an effort, seeking out molecular markers for black rot resistance, while Konstantin’s project does the same with Downy Mildew.

On Tuesday night and Wednesday morning we inoculated a second population, the  progeny of ‘Horizon’ x ‘Illinois 547-1’, with black rot. It went quicker this time due to more help from more people: Bruce, Aaron, and Steve (enjoy your retirement!) were generous enough to donate their evenings to Beth and me.

Grape cluster

So, now that both of our populations are inoculated, we just have to sit back and wait for the disease to take hold. In 2 weeks we expect symptoms will begin to be visible, allowing us to rate individuals in the population for their relative resistance to the disease. In the meantime, however, Beth and  I have decided to start a fun little project, isolating strains of fungi from grapevines throughout Geneva. On Wednesday, we went out to the vineyard and collected a bunch of ugly, diseased leaf samples, surface sterilized them with bleach, and then waited a day for the pycnidia to sporulate.

Unfortunately I didn’t take any pictures of this process, since its all pretty small and we lack a fancy picture-taking microscope, but this video shows how the pycnidia release the spores. It kind of looks like stuff coming out of a pimple, if you’re familiar with that particular brand of grossness, and then I knock off the spores with a needle and plate them on a petri dish.

I used a rig like this, stick-n-poke style. Works well.
I used a rig like this, stick-n-poke style. Works well.

Beth and I had a great time during our leaf collection ooh-ing and ahh-ing in the vineyard about how diseased grapes can become when they’re not sprayed. Here are some pictures of our enjoyment, for your enjoyment.

Beth all up in the vine
Beth all up in the vine
A crunchy leaf
A crunchy leaf
Me all up in the vine
Me all up in the vine
Petiolar lesion from black rot
Petiolar lesion from black rot: eventually will girdle the leaf
Severely stunted and black rot-infected vine
Severely stunted and black rot-infected vine