Weeks 6 and 7: July 13 – 26

I am grouping these weeks together because what I have been doing is a bit repetitive.

On Monday the 13, we harvested the inoculated clusters of V. rupestris B38 x ‘Horizon’ to do incidence ratings. For the incidence ratings, instead of rating on a subjective 0-4 scale, we take a tally of the number of clean/infected/mummified (extremely advanced black rot) berries so that we have an objective, quantitative measurement of disease intensity.  We can therefore do the QTL analysis in two different ways (using either the 0-4 scale or the incidence ratings) to see the differences and similarities that arise between them.

Countin berries
Counting berries

In these two weeks, we rated for disease in ‘Horizon’ x ‘Illinois 547-1’, until Monday the 20th, when we harvested these clusters as well.

On Wednesday the 22, we did our first QTL analysis, on the disease severity ratings from rupestris. We do the analysis using R-QTL, a package in R, which is a code-based open-source statistical software.

The goal of a QTL analysis is to discover regions of the genome that control a trait of interest, in our case black rot resistance/susceptibility. It does this by cross-referencing the segregation of disease intensity (the phenotypic differences) with polymorphisms in the DNA (the genotypic differences). This identifies regions of the genome, Quantitative Trait Loci (QTLs) that are associated with the trait.

Conducting QTL analysis in R
Conducting QTL analysis in R

In our analysis, we discovered two significant QTLs. From these, scientists develop molecular markers, which are short repetitive sequences (SSRs, or microsatellites) located nearby in the genome. An individual’s possession of the SSR, and therefore of the trait, is commonly assessed by PCR (which generates many copies of the SSR as well as of the genome) followed by electrophoresis (which separates the DNA fragments created by the PCR by length). The presence of  DNA fragments the length of the SSR (as diagnosed through the electrophoresis) identifies the individual as having the molecular marker.

Of course, from the SSRs and onward exceeds the scope of my project. My task was to identify the QTLs involved in black rot resistance/susceptibility through phenotyping in the vineyard. The genotyping was done before I arrived, and the development of the microsatellites will be done after I’ve left. Now its left to Beth and future grad students and summer scholars to carry the torch.

Go team Black Rot!

vitisgen_logo

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.

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

Week 3: June 15 – 21

On Monday and Tuesday, I helped Konstantin, a graduate student in Bruce’s lab, perform a detached leaf assay for Downy Mildew, another fungal disease of grapes. Konstantin’s experiment is very similar to mine, in that he is attempting to find QTLs in a mapping population for resistance to disease. However, in my project, we rate for disease susceptibility/resistance in the vineyard, whereas he is rating and inoculating in the lab, where the environment can be more controlled.

The first day, we collected leaves from every individual in the population. The population we used was the progeny of  a cross of ‘Horizon’ x V. cinerea; ‘Horizon’ is a sibling of ‘Cayuga White’, a popular Cornell-produced hybrid winegrape, while Vitis cinerea is a wild american grape variety, with some disease resistance. The second day, we set up the experiment: first we surface sterilized the leaves with a bleach solution, then we cut out 1cm leaf disks and placed them on agar. The next day, Konstantin inoculated each leaf with 50uL of a solution of spores, and returned every day afterward to rate each  individual for resistance based on the prevelance of sporangiaphores (the sporeulating organ of the fungi).

Leaf disks on Agar
Leaf disks on Agar

On Tuesday afternoon, I headed home from work a bit early to make dinner and change into more mosquito-resistant clothes, and arrived back to work at 6pm for evening Black Rot inoculations with Bruce and Beth. It took us about 3 hours to finish inoculating the ‘Horizon’ x V. rupestris population, and the sun was starting to set by the time we finished.

The previous day, we had come out to flag the shoots we were going to inoculate–3 per vine, with 150 vines total–with strips of tape, and Beth sprayed the basal cluster on each shoot with an aerosol-ized  solution of spores. Bruce and I followed behind her, tying plastic bags over the sprayed clusters to maintain humidity overnight, as a humid environment favors infection. This necessitated the evening work, as well as going out at 6am the next morning to remove the bags, as if they were left on in the daytime they would severely limit gas exchange, and choke out the plant.

A plastic bag tied around a flagged and inoculated cluster
A plastic bag tied around a flagged and inoculated cluster

So 8 hours later I rolled out of bed, and Beth and I headed back to the vineyard to pull off bags before the sun got too high in the sky. It only took about an hour, and then we headed home for another hour to shower, or in my case, nap, before coming back in to work. We finished up the day preparing for next week’s similar inoculation of the ‘Horizon’ x ‘Illinois 547-1’ population, and for the rest of the week we worked on preparing inoculum and propagating fungus.

Note: since Bruce and Beth aren’t complete slave drivers, I get to leave early on Friday’s, to make up for working late.

Bonus picture of me in the vineyard looking at Black Rot
Bonus picture of me in the vineyard looking at Black Rot

Week 2: June 8-14

For most of this week, I continued propagating fungus in the lab. However, Thursday the weather was good, so I got to get out in the vineyard to help Bruce’s team prepare to make crosses over the next few days. This work is not directly related to my black rot project, but my other job as a summer scholar is to help out Bruce’s lab in a broader sense, and so I do.

In order to make a controlled cross between two grapevines, we need to assure that no stray pollen can pollinate the flower before we do so. Grapevines have perfect flowers, meaning they have both male parts–the anthers–which produce pollen, and female parts–the pistil–which produce the eggs and eventually will house the developing seeds. On an inflorescence that we will use as a female, therefore, we need to remove all of the anthers from the flowers, in a procedure called emasculation. We emasculate the flowers by removing the anthers from all flowers in a cluster with tweezers, and then putting a paper bag over the cluster to exclude foreign pollen until we can come back a few days later to intentionally pollinate. Closeup of a grape flower.

There are at least as many flowers on an inflorescence as there are grapes on a cluster, and so emasculating each cluster takes us between half an hour and an hour, depending on the size. It’s slow, painstaking, and precise work, but extremely important to do well. After each person finishes a cluster, we have someone else check to make sure we didn’t miss any flowers, because even one anther remaining once we bag the cluster could contaminate the whole thing, and destroy an hours worth of work.

A halfway-emasculated cluster
A halfway-emasculated cluster
A bagged cluster, with initials and date.
A bagged cluster, with initials and date.

Fun fact: we use the same bags to cover the pistilate (emasculated) flower clusters as corn breeders use to cover the tassel and capture pollen: of course, corn is monecious, having the male and female parts on different parts of the same plant, while grapes have both male and female parts on the same flower.

For a great video of the whole crossing process, check out this video by Bruce and VitisGen, where he explains the whole thing.

Week 1: June 1-7

This week, I have been preparing for one of my most important events of the summer: inoculation season! In a bit under 2 weeks, we will be spraying grapevines with a solution of G. bidwellii spores to assess their resistance to black rot in the vineyard.

In the meantime, however, we have to grow a lot of the fungus to have enough spores with which to inoculate. We grow the fungus in petri dishes on agar gel. Our agar is a mix of potato dextrose agar (PDA) and regular agar, to give it both the correct nutrients and consistency. From starter cultures which my mentor, Beth, so slavingly maintains, we remove small chunks of fungus on agar and transfer them to a fresh plate. The fungi then grow to fill the plates, and the cycle repeats. In this way, we propagate the black rot. This work takes place in a sterile hood, which maintains sterility through the laminar flow of air through the hood.

Left: starter culture. Right: propagation culture
Left: starter culture. Right: propagation culture

Our setup in the sterile hood
Our setup in the sterile hood

So thats all I’ve really been up to for the first week, aside from a few trips to the vineyard with Bruce and Beth to check flowering dates.

Week 1: Overview

Back in March, I found out I had been accepted as a Summer Scholar at the New York State Agricultural Experiment Station in Geneva, working with Cornell grape breeder Bruce Reisch. My project’s goal is to investigate the inheritance of black rot disease resistance in grapes. Black rot is one of 3 major fungal diseases (Powdery and Downy Mildew also included) commonly combated by application of synthetic fungicides in conventional vineyards. However, black rot is much more challenging in organically-certified vineyards, as it is poorly controlled by the permissible fungicides (with sulfur and copper active ingredients).

Disease resistance is very important, both to decrease fungicide use in conventional vineyards, and to facilitate successful cultivation of organic vineyards. Personally, I avoid organic fruits, because I know most are sprayed excessively with copper, and are often more damaging to the environment than their conventionally-grown counterparts. This article is a good overview of the issue, for those interested. Decreasing pesticide use by utilizing disease resistant varieties makes grape growing safer, easier, cheaper, and more environmentally sustainable.

So, that’s a quick overview of the scope of my project. The rest of this blog presumably will document what I do on a weekly basis. If that interests you for whatever reason, anonymous internet reader, do read on.