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Australian Macadamia Society Podcast: Episode 4

In this AMS podcast, AMS industry development manager Leoni Kojetin chats to Helen Wallace, Professor of Agricultural Ecology at Griffith University in Queensland.

Professor Wallace has been a macadamia researcher for more than 20 years and is part of a team of researchers investigating pollination in a number of crops including macadamia. The research is funded by the Hort Frontiers Pollination Fund and has shown that better pollination has the potential to improve yields and increase kernel recovery. Professor Wallace summarises the major findings to date, explains the latest paternity testing techniques (mass array and genotype by sequencing), outlines recent work on large monovarietal blocks, and details a large experiment that added 1 tonne per hectare with good pollination.

EPISODE 4: Pollination in macadamias – the latest research

Leoni Kojetin (Australian Macadamia Society). Today we're chatting with Helen Wallace, Professor in Agricultural Ecology at Griffith University. Helen has been involved in macadamia research for many years and is part of a team currently investigating pollination in a number of crops, including macadamia. Welcome Helen.

Helen, who is doing this pollination research that you're involved in?

Professor Helen Wallace (Griffith University). We've got a team of researchers at Griffith Uni, including Dr Wiebke Kämper, Anushika De Silva and Joel Nichols. The project is led by Professor Stephen Trueman and funded by the Hort Frontiers Pollination Fund.

Leoni. From your experience with many other crops that you're working in pollination, do you believe that macadamias are under pollinated?

Helen. Yes, absolutely. Based on our recent research, I do think that they are under pollinated.

Leoni. Getting back to basics, do we know what actually pollinates macadamia?

Helen.  Yes, we've known this for many, many years. We know that bees are good pollinators of macadamia. Both honeybees and stingless bees can act as pollinators. We know that there are lots of other insects that might also act as pollinators; in the wild, it might be things like birds and bats.

Leoni. During full flowering there seems to be a lot of pollen floating around in the orchard. Is wind assisting with some of this pollen movement?

Helen. This is something that a lot of people tell me. 

One thing we know about macadamias is they're not adapted for wind pollination so, for example, they have nectar. Plants that have nectar are usually trying to attract animals to come and take it. We also know that they have sticky pollen that forms in clumps and tends to fall to the ground. It might be that there are a lot of pollen grains floating around, but a wind-pollinated plant typically will have a large surface to catch the pollen, and we know that macadamias don't have this. They have an extremely tiny surface, smaller than a pin head, to catch the pollen. So, given all those things, they're not adapted to catch the pollen.

That doesn't mean that wind pollination doesn't happen occasionally. It might happen because at flowering time, as we all know, there's a lot of pollen floating around, and it might accidentally land on the stigma. But if it does, it might be self-pollen and it might be dead pollen that's been floating around for a few days.

Leoni. So, pollen has a certain viability and can die?

Helen. That's right. When pollen is freshly released, it's usually the best pollen for the first probably eight to 10 hours after it's officially released. We don't really know in macadamias, but in other plants, pollen can maybe only last a few hours; in some plants that might last a few days. In macadamias, we don't really know but there is a good chance, if it's been floating around in the atmosphere for three, four, five days, it might no longer be viable. So, it can't grow and it can't fertilise the flower.

Leoni. Can you explain the difference between self-pollen and cross-pollen?

Helen.  Again, a lot of growers have asked me is cross-pollen from a different tree or from a different flower. Cross-pollen is any pollen from a different variety and self-pollen is any pollen from the same variety. For example, if you get pollen from a 344 and you go and get pollen from 344 in California or in Hawaii or in South Africa, that is still self-pollen. It doesn't matter where it comes from, it's all about the genetics of that pollen. If that pollen is the same as the parent tree that you're putting it on, the same variety, that is self-pollen and cross-pollen is any pollen from a different variety.

Leoni. From the research, what do we understand about macadamia's need for this cross-pollination?

Helen. We've got a lot of new research on that, but I probably need to go back to basics and explain a little bit about inbreeding. 

Macadamias are plants and they don't like inbreeding, just the same as animals. We all know what happens when you inbreed animals or inbreed humans. We know that means that the offspring are likely to be deformed or they're likely to be weak and small and have all kinds of problems so they may not survive. It's exactly the same for plants. 

Macadamias in the rainforest have evolved to stop inbreeding and they've got all these mechanisms that they have that stopped them from breeding with themselves. The funny thing about a macadamia is that, in animals we have male and females and they're separate, whereas in macadamia, the male and female bits are right next to each other. This means they're going to accidentally self-pollinate all the time and that's a really bad thing if you're a macadamia, because it means you're going to have deformed small and weak offspring.

Over millions and millions of years, macadamias have evolved to stop that from happening, like many tree crops, and the way that they do that is they chemically recognise pollen that's self-pollen and they stop it from growing down. They don't want to waste any of their effort making a nut that's going to be small and weak and unlikely to survive. 

So that's the first problem. They've got a chemical system that stops the self-pollen from growing. If the self-pollen does make it, which it sometimes will, you've got an embryo that's going to be small and weak and not as strong as those from cross-pollination. That's why macadamias need cross-pollination - they've evolved both to prevent self-pollination and to try to make sure that they're having as healthy offspring as they possibly can.

Leoni. So, cross-pollination is important then for nut set. What do we know about the longevity through to the final crop though, because many growers do have those mono-varietals blocks?

Helen. That's a good question and that is something we've been wondering about for many, many years, but we've been able to resolve that with recent research using paternity testing of the crop at harvest.

Leoni. When you say paternity testing, can you give us an explanation?

Helen. Okay. So, the nut, the kernel, that's the baby, and we want to find out who the daddy is. As with people and animals, we want to find out where did the pollen come from that made that embryo. If you have a mother and you have a range of different fathers, you can figure out who the father was, and we've got really good genetic techniques that can figure this out now.

There was some early work in the late ‘90s that showed that A16 was around about 90% cross-pollinated, even in the middle of a really large block, which was really surprising, and this work showed that the yield dropped off as you moved away from the other variety. Now that we've got these big, new, fantastic paternity testing techniques, we were able to go back and revisit some of those large blocks and find out who the father was for all the nuts. The techniques that we used were mass array and genotype by sequencing and we looked at what was happening in great big blocks of 816 and Daddow.

Leoni. Can you explain the mass array in genotype by sequencing briefly for us lay people?

Helen. In the old days we had to do tests on the nuts where we got a whole lot of markers and we put it all together to figure out who the father was. These days we can get big, long sequences of DNA from the nuts and then we can try to match that to the parents. Because we've got a lot more data than we used to have, it's much easier to do this. Once we've got a library where we can tell, okay, this is what 344 looks like, this is what 816 looks like, this is what Daddow looks like. We can then go back and match those sequences to the nuts to find out who the likely father is.

Leoni. Can you tell us about recent work that you've done on some of those big mono-varietal blocks?

Helen. We went into some blocks - there were about 48 rows of Daddow and 48 rows of 816 - and we were expecting a lot of cross pollination on the edges of those blocks where the Daddow is next to the 816, but we thought we'd find a lot of self nuts right in the middle of those blocks. 

We went into row 23, right in the middle of the block as far away from the other variety as we could, and we paternity tested all the nuts in there along with all the nuts on the edge. We thought we'd find a lot of crosses at the edge and maybe mostly selfs in the middle. Much to our surprise, we found 90% of the nuts were cross-pollinated even in the middle of the blocks and it didn't drop off as we moved away from the cross-pollen, so right in the middle of those great, big, pure blocks, there were hardly any selfs at all. They were all cross-pollinated and that was a big surprise to us.

Leoni. If such a high percentage of nuts are cross-pollinated, does that mean that obviously we have adequate pollination and we don't need to manage it more intensely?

Helen. That's one explanation, but there's another explanation, which is a really important one for growers to think about because it might impact on the yield. 

Another explanation is maybe they've set lots of crosses, but they can't hold their selfs under any circumstances. So, if there's some really strong barrier to macadamias hanging onto their selfs and turning them into nuts, you might find lots of crosses and you might never find selfs even in the middle of a big block. But if this is true, we might find that the yields in those big blocks are lower than the yields on the edge of the block because those trees in the middle of that big block are missing out on lots of crop because they just don't get enough cross-pollen in there.

In the middle of the big block, we thought we might get one or two tonnes per hectare of crosses, but on the edges where there are more bees or there's more cross-pollen, you might get four tonnes per hectare of crosses and that would be another way of looking at it. So, either they're really well pollinated or you're missing out on a lot of crop in the middle of that block where you haven't got enough cross-pollen.

Leoni. Do we know more accurately how much yield is potentially lost from this poor pollination?

Helen. We do now and it's something that I've been talking about with other researchers for 30 years and we finally got the chance to do the big experiment that we've been dreaming of for a very long time. 

We got whole trees of Daddow and 816 in those big blocks that I was talking about and we pollinated as many racemes as we possibly could to see how much more yield we could get if we had really good pollination as a way of finding out what's the carrying capacity of the trees. In other words, if you throw all the pollen in the world that you've got at it, what's the maximum yield that we can get from those trees and to find out if we could lift the yield from what it was doing at the moment? Now, this is a crazy experiment because we had 10 trees crossed and 10 trees as controls in each row that we did it.

We did one row of Daddow next to the 816 and we did one row of Daddow in the middle of the block. We did the same for 816, one row of 816 next to the Daddow and one row of 816 in the middle of the big block. 

Now we have to do replicates and controls because we have to do a proper experiment so we had about 10 trees crossed and 10 control trees in each of the rows and we hand-pollinated every raceme we could on those trees where we cross-pollinated. It took a lot of people a lot of hours. We had a team of 10 people working all the time in the orchard and, in the end, we managed to hand-pollinate about 40,000 racemes all up to do the experiment.

That was exciting for me because it's something that we never knew. What is the carrying capacity of the trees? How much can we get out of these trees if they've got all the pollen in the world thrown at them? For 816 we lifted nut-in-a-shell yield from 1.3 to 2.5 tonnes per hectare in the middle of the block, a massive increase. For Daddow, in the middle of the block, we lifted it from 2.8 to 3.9 tonnes per hectare, a very big increase in yield. Even on the edge of the blocks, right next to the other variety where you think pollination was really good, we still managed to lift nut-in-a-shell yield by about 30%.

For 816 it went from 2.1 to 2.7 tonnes and for Daddow, it went from 3.1 to 4.0 tonnes per hectare. So, by throwing all the pollen in the world that we could, or the cross-pollen in the world we could at these trees, we managed to lift the yield by about a tonne per hectare. So, we think that that's how much the macadamias are under pollinated.

Leoni. How then do you explain that many growers have a big block of the same variety and still get good nut set?

Helen. That's a good question that we wondered ourselves and we've got three possible reasons for this. 

It might be that the pollen is coming from a long way. When we did our paternity testing, we found that some pollen was coming from more than one kilometre away so pollen can be carried by bees a very long distance. 

Another reason for why we might get nut set in these big, pure blocks is that the block may not be just one cultivar. Every grower that you speak to knows about ring-ins - there are always some varieties that aren't what they were supposed to be or that don't actually look right and that might be because there's been a rootstock that's grown up. Or it might be because one variety somehow got mixed up with other varieties or it might be trees that get replaced after storm damage.

This might happen also in the nursery, where one variety gets accidentally swapped with another one, or it might happen at planting. We don't really know. But we do know it's really common to have ring-ins in an orchard. Everyone says they've got a pure block, but every grower that I know can go down and say, well, that’s not actually a 344, that's something else. Well, that's not this, it's actually something else and there are ring-ins that you can see, but there are also the ring-ins that you can't see as some varieties are really hard to tell apart. 

The result is that instead of having a pure block, you've got a mostly pure block with a few trees here and there that are not that same variety, that are different variety. The good thing about that is that this might be why we're getting some set in those really big, pure blocks.

So that's our second reason, that was a very long explanation. 

There's a possible third reason and there's also some evidence for this. There may be some varieties that are self-fertile. There's some good evidence from Catherine Nock's group at Southern Cross University that 741 can set selfs. But then the next question we have about those varieties that can set self is how does that affect kernel recovery? 

The handful of selfs that we got from 816 had a kernel recovery of about 30% compared with 44% for the crosses and it goes back to that thing that I was saying before about inbred things are weak and small and not very strong. We don't really know if nuts that are selfs are actually going to be smaller than other ones.

Leoni. Helen, you mentioned 741 being a possible self-fertile variety. Are there any other varieties that we have that could be self-fertile?

Helen. That's exactly what we want to know and we're in the middle of doing paternity testing of about 20 varieties to find out which ones are self-fertile. We want to find out for these varieties which ones are self-fertile. Just because they're self-fertile doesn't mean that the whole crop will come from selfs. It might be that they're self-fertile, but only 30% of the crop comes from selfs. But we also want to know which pollen parents make big nuts and which pollen parents make small nuts. So again, back to that thing about inbreeding. 

Think about a dog for example. If you cross a big dog with a small dog, we know that the puppies are going to be bigger. We think that the same thing is going to happen with the nuts. We know that different cross-pollen parents might be able to produce bigger nuts or smaller nuts but, most importantly for growers, we want to know how pollen parent affects kernel recovery. As I mentioned before, we know that selfs tend to be smaller and have lower kernel recovery, but we want to find out for all these varieties, what's the effect of pollen parent on kernel recovery.

Leoni. Your research has clearly shown a potential for a yield increase and a kernel recovery increase from better pollination. What about other quality attributes linked to cross-pollination?

Helen. We've got some work ongoing on that, but I can't tell you the answers to that yet. We're still working through the data, but that might be sometime next year.

Leoni. Do you believe that improved pollination potentially increases youth and quality in orchards in all settings or just in those settings that are under pollinated?

Helen. That's a really interesting question. The fact that we found that trees in the middle of large blocks are under pollinated by about 1.1 to 1.2 tonnes per hectare was really surprising to us. But we're also finding that the trees right next to another variety still were under pollinated by about 0.7 to 0.9 tonnes per hectare. Now, we haven't looked at this on very many orchards so we don't really know what's going on out there in the industry as a whole, but it probably indicates to us that under pollination might be much more widespread than we thought. 

So, hard to know but one of the things that growers can do is to check for clumps of pollen on their flowers in the early afternoon. That would mean there have been no bees there. If they've got really low initial nut set at about three weeks after flowering, that might mean that they've got a pollination problem. If they find that their yield declines as they move away from another cultivar, that might mean that they've got a pollination problem, but we really don't know.

Leoni. Talking more about this average grower, what does this mean for them in terms of orchard design potentially or management of orchards into the future?

Helen. I think for any grower putting in new orchards, they should seriously think about inter-planting cultivars. If it's a grove and established orchard, they might think about replacing storm-damaged trees with another cultivar or about ways to get the pollen different varieties close together. It's really important to make the job as easy as possible for the honeybees by putting the cultivators close together and hopefully that will help to get the cross-pollen in. 

The other important thing is to bring the honeybees in and to spread them around the orchard as much as possible. Also, try to avoid using any pesticides during flowering and make sure that they're not going to hurt the bees if they're going on to the crop.

Leoni. In conclusion, what is next in our understanding about pollination in macadamia?

Helen. We could probably learn a lot from almonds. In almonds they have known self-sterile and self-fertile varieties and they strongly interplant the varieties. So, they have one variety planted next to another variety, in alternate rows, i.e. variety one, variety two, variety one, variety two. They actively manage their bees so much so that there are pollination contracts. There are projects on trying to find ways to make sure the bees have proper nutrition when they go onto the farm and they do everything possible to try to get those pollinators in there. 

I think that they are the sorts of things that the macadamia industry should think about as ways to manage this and solve some of these issues.

Leoni. Thanks, Helen, that was really interesting and certainly informative. We look forward to catching up when you have even more updates on the research that you're doing. 

For our listeners, if you want more information about pollination in macadamia, Helen and her team have authored several macadamia News Bulletin articles which are available on the AMS website, and there is more information on the Hort Frontiers Pollination Fund website. Lastly, we've published a new AMS fact sheet on pollination and that also is available on the AMS website. Thanks for your time today Helen, we appreciate it.