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
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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.