El laboratorio

Yesterday, not much special happened. Philippe and I both used the spare time in the morning to work on some things that we did have time for to do whilst on the road. The afternoon we spent waiting at the airport and the evening predominantly in a taxi, Evening traffic was, well, let’s say, dense. It took us more almost 3 hours to travel the 20 or so kilometers that lie between the airport and our hotel near the CIP.

Today was definitely way more exciting. When Philippe went his way to check on his possible Ralstonia strains, I went to the Phytopthora lab to get updated. As with any research project and probably even more with these kind of exploring projects there was good and bad news.

Philippe had quite a meager score, only six or seven isolated bacteria from the samples. However, this makes sense in many ways. Many stem samples were quite dry and the plants were not wilted, so the bacteria are probably not very heavily present. It could also be that the methods used were not effective enough to extract the bacteria from the sometimes even woody sample and lastly it could very well be that these particular Ralstonia strains are viable but not culturable or maybe they are culturable, but on slightly different medium than the known Ralstonia from cultivated tomato. DNA test will now have to prove if and how many plants really contained Ralstonia and what these isolated samples exactly are. Pretty exciting!

I have learned that the isolation method with the sliced potatoes (which works well in the lab for Phytophthora strains from potato), does not seem to work at all. Nothing grew. This could be the way the potatoes had been handled and stored on our route. But, we cannot bring 18 degrees high humidity incubators during the trip, so unless some very different results come from this weeks samples, we don’t have to use this method again. Somehow that’s a shame, because carrying petri dishes around is way more hassle and also way more prone to contamination. One day last week, I had to throw half the petri dishes out, because they were contaminated during our travels. That meant that day, I could only use the potato method to store samples. Then again, P. infestans grows slow and there is still a chance that some mycelium appears on the tubers. But, I guess this is already a lesson learned: Bring more and cleaner petri dishes!

On the other hand, the technicians had seen actually  numerous good looking symptoms on quite a lot of the leaves that we collected the beginning of last week. The bad news was that even though the symptoms were there, is was very hard for them to recover live Phytophthora from the leaves, because they were so dry. I think this is partly due to the used methods,which differ a lot from the methods we use in the lab. Here they wait for the leave to spontaneously sporulate and wash off the spores and sporangia and then look for them in the washed off watetr. This is highly diluted and often contaminated with other sores, so it’s not an easy task. In Freising we would force Phytophthora to grow directly on a plate with rich medium and only later we worry about how to get the isolate clean. This is something that I should have checked beforehand. These are not cultivated potatoes, so we might have fewer spores to start with and even with potato, they apparently have problems here every now and again, especially when it has been very dry. And it has been dryer than it should have been at this time of year.
Later in the day,I had a look at the some of the samples from the second half of last week and I’ve seen Phytophthora sporangia washed of from both S. pennelli and S. peruvianum. The samples from this week we only just brought in, so nothing can be said about them yet, so it’s looking quite well at in the end.

But the fact that so many samples from the first few days were negative, didn’t feel well to me. In my mind, rescuing Phytopthora from a leave shouldn’t be so hard and the symptoms were evident, so I was not really prepared to use molecular detection methods. Luckily, a few days ago, I came across a paper with a brand new DNA extraction method.  This seemed like a good excuse to test this method and use molecular methods to detect the presence of Phytophthora in the leaves. We tried the method on 24 samples, including mycelium freshly scraped from a potato. By Monday we should have the results. If the results are negative, there are several other methods that we still can test and there is a whole batch of samples that have yet to be processed and I will also be bringing FTA samples with me to Germany.

To me, this trip was definitely a success. I have seen sporangia directly coming of samples from two different tomato species and sporangia from a third third species were prepared by the technicians and almost half of the samples are still waiting to be processed. The molecular results will tell us how much we might have missed. To be fully prepared for the next trips I will compare both isolation methods one on one when I’m back in Freising. I also have to work a bit on my route planning. Being almost without petrol or underestimating the driving time by about 4 hours are not very clever, but easy to solve!

I am fairly sure that most readers of this blog are predominantly here to look at the pictures of the tomatoes. So, I will not post any more updates on the results. If you are interested to hear more of my experiences and the  outcomes, please do get in touch via email. If you want to see more pictures, I invite you to check this website again some time from February next year, when Philippe and I will go to the north of Peru to look for a few other tomato species in slightly wetter climates.

Gracias por leer mi blog y hasta la próxima!

Cip

Yesterday we had a break from the field work. It was still a pretty busy day. Working days at CIP start early and both Philippe and I wanted to have a look in the labs and see some of the first results.

After breakfast we started with a seminar, Jean Ristaino talked about her work on global P. infestans diversity, which was very interesting. After the seminar, I could have a quick look in the lab, where two of the technicians could show me the plates with the results from our first day collecting. One of the reasons for this trip was to figure out what the best sampling methods are. We can take the infected leaves and put them in a petri dish with some water agar to keep them humid, so that they don’t dry out, but carrying loads of plates is not easy and in the lab, you can get any kind of growth on these plates, the pathogen that you want, but also opportunists that make it harder to isolate the real infecting pathogen Moreover, plates get contaminated when not store properly and I had already thrown away some plates the previous days. Alternative methods include using sliced potatoes. You can store leaves in between two slices and keep it for a few days. The idea is that the potato then gets infected and that after you are back in the lab you can grow Phytophthora on the potato and isolate it later. This is supposed to be cleaner and the potatoes are easier to carry and obtain, but we don’t know if it works with at all Phytophthora strains that grow on wild tomatoes. They might not be adapted to growing on a potato. Another issue might be being away from a temperature controlled lab for 3 days. That might stress the sample so that we cannot recover anything. Unfortunately, both methods take mostly more than 5 days to get proper results. So, so far we have only one P. infestans sample that has already been processed further. That means, the spores of P. infestans had grown, were washed off and reinoculated on fresh, susceptible potato and tomato leaves. The cool thing is, it came from the first plant collected on the first day. Besides that there are a large number of promising candidates from the first three days, but also a back-log of samples that we brought in yesterday. Manpower is limited, but we can store the samples in a cold place for a while and process after the weekend and a lot of boxes with sliced up potatoes are already prepared and filling the lab.

At 11, I had a meeting with Tiina Sarkinen. She is an expert on wild Solanum species, including tomato, and it was really interesting to hear her thoughts and to share experiences.  Of course, I already knew that there are a lot of interesting wild solanum species, but that there were that many. There is definitely room for a lot more research in this area.

For lunch we went to a nice little restaurant on the campus of the nearby national agricultural university La Molina. I don’t say it often, but the portion was way too big. Very tasty though. Anticuchos served with typical Peruvian potato and maize varieties. Tiina had recommended me to take a little walk on the campus after lunch. I’d find some nice things there, and she was right. Next to some of the trial fields, there was a good population of S. pimpinellifolium. Some were clearly suffering from one thing or the other, but other looked beautifully healthy. I collected three specimens of which one is almost certainly infected with Phytophthora. Now that is easy sampling.

Philippe has spend part of the day in the lab looking at his own samples. For him this trip is not so much about how to sample and process Ralstonia, he’s been doing it for years. For him it is the question whether there is Ralstonia at all in these environments and in these species. If there is, that might explain more about the evolution of the species. Also he is not alone in the lab.

When I returned at the CIP, we had to pack our stuff for the next week. Double checking if we still have all that we needed. From pens to plastic bags, but also letters that explain what we do, so that we don’t get in trouble with the police. We had to hand back the truck we rented and sort the paperwork for the car rental and all other things we do next week. As one would expect, these things take forever, so by the time this was all done, we had to get ready to go for dinner. Dinner was at a house warming party of some people working at CIP. We felt very welcome in the CIP family and it was very nice to talk to all people there and to hear about the different research going at the institute.

Today, I will make the planning for the rest of the stay here and I also hope to have a quick look in the lab in the morning, if someone can let me in. The afternoon will be used to work away all unanswered emails and other things from the past few days. Late afternoon we fly to Arequipa for the last leg of this trip. I am really looking forward to that. This is the area where S. peruvianum and S. chilense grow. S. chilense are the plants that I have worked with in Freising for the last 2 years.
I must also say, it is very exciting and rewarding to do this work, but also really tiring. So, before we hit the road on Monday again, we will use the Sunday to relax in Arequipa and be tourist for one day!