Azolla, the wonder R. L. Bieleski1

It was a bit prophetic that the very first branching thread, with leaves and colour photo I ever took, number 0001 roots coming off at the nodes. The in my collection, taken with my trusty small overlapping scale-like leaves Kodak Retina camera and a spectacle have large air spaces, giving the lens, was a close-up of plant its buoyancy. Reproduction is rubra at Bethell’s Beach, Auckland. largely vegetative, with side branches Prophetic, because 25 years later breaking off from the main plant and I was to spend 9 months on study growing to form a new plant, from leave at the University of , Fig. 1 Individual of Azolla rubra. which further side branches split Davis, probing the physiology of this Photo: Murray Dawson. off, and so on. What this means in fascinating plant. But before I get to effect is that given ideal conditions, my own small contributions I want to Azolla has an exponential growth take you for a small tour highlighting pattern, and we can talk about its some of the things we know about doubling time as a measure of its this remarkable plant. growth rate. This is not a theoretical concept: in my experiments I grew First, the . Despite the Azolla mexicana under near-optimal small size of an individual plant, conditions, and exponential growth 1–2 cm across (Fig. 1–2), and its was maintained over five generations, habitat, floating on the top of ponds Fig. 2 Close-up of a dense Azolla rubra mat, till space in the growth tubs became (Fig. 3–4) usually in company with growing in a pond at Halswell Quarry Park, Christchurch. Photo: Murray Dawson. limiting, causing crowding of the the monocotyledenous Lemna fronds and therefore limiting the (duckweed, ducksmeat), Azolla exposure of individual fronds to light. is actually a in its own family, The original 500 mg starting weight Azollaceae, though current research of inoculum consistently reached now places it in the ca. 16 g in 11–12 days, giving a together with another floating fern, doubling time of 2.3 days. Salvinia. Azolla has a worldwide distribution in warm, subtropical and In one stage my growing went tropical regions. Taxonomist’s views through three iterations, meaning vary a little, but most centre about Fig. 3 Azolla rubra plants (red) competing that after the 12-day period I took there being six species worldwide with Lemna minor plants (green) in a rāupo 0.5 g samples from that 16 g to seed wetland, Flaxmere. Photo: Rod Bieleski. (Fig. 5), with the scientific name for new tubs, and then again 12 days ’s single native species later, with exponential growth being having done a taxonomic do-si-do maintained right through. If each time from A. rubra through A. filiculoides I had been able to use the full amount and A. filiculoides var. rubra and in seeding tubs, I would have had 32 back to A. rubra (its main Māori tubs at the second iteration and 1024 name is kārearea). To complicate at the third, with a final tissue weight things in New Zealand, in recent around 16 kg. In the field, comparable years an exotic species, A. pinnata, growth rates are sometimes reached, has come into our country and has Fig. 4 Azolla rubra growing in a drainage as doubling times are often spoken of been taking over from our A. rubra in channel alongside Pegasus Bay Walkway, as being as low as 3–5 days. It’s hard areas north of Auckland. The jury is north of Christchurch. to comprehend the power of such Photo: Murray Dawson. out as to whether A. pinnata entered growth, so can I pose a question for naturally (such as on waterbirds’ you? Let’s begin with my standard feet) or accidentally through human inoculum, 500 mg of Azolla, about actions, but it has enjoyed our climate as much as will fit on the end of a and waterways enough to become teaspoon. If we were to allow that classed as an invasive species. Azolla to keep up its exponential growth without limitations of light, The typical fern structure has nutrients and space coming into play, been modified in Azolla to a point how long would it take for the weight where the stem and rhizome have Fig. 5 Azolla on Lake Macquarie of our Azolla crop to equal the entire become reduced to a frequently- (NSW, ). Photo: Rod Bieleski.

1 33 William Bond Street, Stanley Point, Auckland 0624, New Zealand; [email protected]

New Zealand Garden Journal, 2015, Vol. 18(2) 5 plant biomass of the whole world? years ago when they were already biological fertiliser, and its use on rice A million years? A thousand years? using it as a nitrogen fertiliser for their may be declining. However, uses as A century? All wrong. It would take rice paddies (Fig. 6). The earliest supplements for animal and bird feed about 5 months for the doubling known written record of the practice is are being explored and expanded, time of 2.3 days, or 9 months for a in a book by Jia Si Xue in 540 A.D. on and small lots of Azolla growing in doubling time of 4 days. Of course The Art of Feeding the People. Its use canals and ponds as food for pigs and limitations do occur, saving us from in Vietnam dates to the 11th century. ducks are now ubiquitous throughout being buried in Azolla, but this does By the early 17th century, its use as southern China. A factor in the use explain why a pond almost free a compost was being documented in of Azolla as a green manure and of Azolla can become completely many local Chinese records. an animal feed is its low carbon to covered in less than a month. nitrogen ratio of about 10:1, meaning that when used as a green fertiliser its Though the growth is primarily nitrogen supply cannot be swamped vegetative, Azolla can behave like by bacterial activity. As an animal more typical and reproduce feed, it has a protein content between sexually, but it does have to do it in its 13 to 24% on a dry weight basis, own way. The standard fern way is for making it a very high quality plant the diploid generation, the sporophyte protein source. that we recognise as our fern, to shed haploid spores into the wide world, So where did I come into the act? At there to germinate into separate the time, in 1979, Professor D. W. life stages, the haploid prothalli Rains of the Department of Agronomy (small liverwort-like plants) which and Range Science (University of produce the eggs to be fertilised by California, Davis) was heading a large the swimming sperm, creating the program trying to understand the new diploid (sporophyte) generation. biology of Azolla, with a view to using With Azolla, spores are not released; it more skilfully and further expanding instead male and female sporocarps its agricultural uses. A problem that are formed on the underside of Fig. 6 Chinese farmer inoculating a rice showed up was that Azolla seemed the frond, held there, and the paddy with Azolla 1500 years ago. to have a high requirement for fertilisation occurs on the parent Under field conditions Azolla can phosphorus, with phosphorus supply diploid sporophyte. In this respect, accumulate up to 2–4 kilograms often limiting growth. A typical finding where there is no physically separate of nitrogen per hectare per day, was that even when supplemental gameophyte generation, the life cycle 1.1 tonnes of nitrogen per hectare phosphorus was provided, less resembles that of higher plants! per year, and almost three times than 20% was actually utilised by the Azolla, so that even though the But that is not what makes Azolla the performance of legumes such in-plant N:P ratio was around 10, the really special, it is its ability to fix as clover at around 400 kg of yield of N fixed to P supplied was nitrogen, which it achieves by nitrogen per hectare per year. It is more like 1:1 to 2:1. As an old hand supporting colonies of blue green no wonder, therefore, that use of at studying phosphorus nutrition of algae (cyanobacteria), Anabaena Azolla has become a major tool in plants, I was given the job of trying azollae, living in the air chambers of the growing of rice in China and to uncover some basic facts about its fronds. And it is an association Vietnam. A major push for expanding phosphate transport and utilisation in that puts all other nitrogen-fixing the use of Azolla began in those Azolla, to see if ferns were “different” systems to shame. The growth two countries in the early 1960s. In to the higher plants I had worked on rates I reported, of a doubling time 1980, Azolla was being grown as a up till then. The basic setup I had of 2.3 days, were achieved in the green manure on about 1.3 million available in which to grow my plants absence of any nitrogen supply in hectares of rice in China alone. In was a growth cabinet which gave the nutrient medium that the Azolla the paddy fields, the typical pattern near-optimal conditions for Azolla: was growing on. Every last skerrick is for some of the Azolla to die, as 27oC, 16 hour day, about 25% of of the nitrogen was fixed by the the mat becomes fully shaded by full daylight, standard plant nutrient Anabaena and swapped to Azolla in the rice, sink and decompose – its solution minus any nitrogen source, the form of NH + (ammonium), with rapid decomposition means that 4 in 14 cm × 14 cm × 20 cm plastic the Azolla providing Anabaena with its nitrogen and other nutrients are tubs, with aeration. Movement of carbohydrate, mainly as fructose. made available for uptake by rice phosphate in experimental tests was At one stage I got curious as to how during grain development. Besides followed using the radioisotope P32. much faster Azolla could grow if it this straightforward contribution as If you want to know the gory details, didn’t have to rely on Anabaena for a nitrogen fertiliser, Azolla is also see Bieleski and Läuchli (1992). its nitrogen and so I supplied it in a important after rice harvesting in normal growth medium. The answer? trapping nutrients out of the water The most basic thing I found out Growth was actually inhibited a little that might otherwise be washed about Azolla was that its ability to bit! The consequences of this ability away and also in smothering weed accumulate phosphate was not in to fix nitrogen were recognised by (and mosquito) growth. I have had any way inferior to that found in Chinese rice farmers at least 1500 trouble finding out its current use as a higher plants. Rates depended on

6 New Zealand Garden Journal, 2015, Vol. 18(2) whether the plants were being grown non-living and structural tissues in What of the future? From what I have in an ample supply of phosphate Azolla. No wood, damn all xylem, it’s found, we can suggest ways to attack (0.2 mmolar) or were put into a all living and active tissue, and the the problem of phosphate shortage phosphate starvation regime, when concentrations are not at all high for in the life of Azolla. We could breed the plants developed an additional young plant tissues. Instead, I feel Azolla with very much longer roots, or “low phosphate accumulation that the reason for the sensitivity of decrease the water level of paddies system” and were able to accumulate Azolla to phosphate supply can be to a point where the Azolla roots phosphate about two times faster. found in the relationships between the can access the soil. Neither seems Under the starvation regime, plant, the water in which it is growing, practical. Instead of putting phosphate Azolla was still able to show a net and the soil of the pond beneath. In a into the paddy in a single dressing, uptake of phosphate when the paddy field, the water may be 15 cm we could dribble it in over the growing external concentration fell as low deep and contain 5 mmoles per season – a bit complicated, but as 0.1 μmolar, 1/2000th of that in cubic metre of inorganic phosphorus doable. Or we could aerate the pond, the growth solution and 1/30th that in solution at the time of inoculating stirring the water and breaking down in typical soil water. The phosphate with Azolla. It means that it can only the diffusion barrier between soil pump was very powerful: the support growth of about 10 g fresh and water. Or we could go further concentration gradient between the weight per square metre before the and agitate the surface of the pond environment supplying the phosphate water becomes completely depleted soil to also increase the ease of at 0.1 μmolar and the tissue content, of phosphate, whereas a dense transfer of its bound phosphate into 10 mmolar, was 1:100,000. To put Azolla cover is more like 200 g the pond water. And will Azolla have it in context, 0.1 μmolar phosphate fresh weight per square metre. For a developing future in agriculture, or is what you would get by dissolving further growth, phosphate has to will its use fade out with the decline a heaped tablespoon of sodium be supplied from elsewhere – i.e., in peasant farming in India, Vietnam phosphate in an Olympic swimming released by the soil. The nature of the and China? I hope not. At present, pool, and 10 mmolar by dissolving relationship between the soil minerals almost all nitrogen fertiliser used in that heaped tablespoon in a large and inorganic phosphate is such that agriculture comes from the Haber bucket. The next question I attacked the phosphate is strongly bound, process, which is very energy- was “how much of the phosphate is to the point that the equilibrium intensive. In total, global Haber taken up by the roots, and how much concentration with the surrounding ammonia synthesis uses of the order by the fronds?” The roots were about soil water is typically around 3 μmolar. of 1.5% of the world’s annual energy three times as efficient as the fronds, That is the concentration that typical supply. So as we move towards on average, at taking up phosphate. plant roots face (and explains why reducing our use of energy in a drive However there was a greater the phosphate accumulation pumps to slow global warming, Azolla offers proportion of frond tissue, so that are so effective). With plants growing a small lifeline (Fig. 7A–B). the total uptake into the plant carried in soil, the root hairs are never more out by the fronds themselves ranged than a millimetre or two away from from 70% of the total when phosphate the phosphate-binding soil particles. was in abundant supply (100 μm) to But Azolla roots do not penetrate the 45% when phosphate was limiting soil and are not in close contact with (1 μmolar) and at a concentration the soil particles. Thus, for much of more typical of field conditions. its growth period the Azolla crop must Nonetheless, all the uptake studies be completely dependent on diffusive said that Azolla was very efficient transfer of inorganic phosphate at extracting phosphate from the from the paddy soil particles to the water, and its tissue content was not adjacent water, but then through a A unusual, and so the reason for the 15 cm layer of water to the root and apparent high demand for phosphate frond surfaces. This can be expected had to be sought elsewhere. to be a major rate-limiting process (Bieleski and Ferguson, 1983). A root So let’s look at the field situation. system that penetrates the soil offers Maximum field growth rates of Azolla a much more effective interface for are usually obtained only after heavily phosphate transfer than the thick fertilising paddies with phosphate, and layer of water that the Azolla must this is what has led to suggestions depend upon. It has been noted that that Azolla may have a high specific a water level which allows Azolla phosphorus requirement to support roots to touch the soil will often cause growth and nitrogen fixation. Though phosphorus deficiency to disappear B the phosphorus content of Azolla at (Lumpkin and Plucknett, 1980). So 0.3–0.6% dry weight (5–10 μmoles Fig. 7A–B Research appears to have there it is: the Azolla is phosphate- continued at an Azolla production and phosphorus per g fresh weight), is a limited not because of its physiology, demonstration farm at the Philippine Rice bit higher than the normal values for Research Institute (Muñoz, Nueva Ecija). but simply because it is floating free most plants, 0.2–0.25% dry weight, Photos: “Judgefloro” (CC-BY-SA-4.0). on top of the pond, and so its roots that difference can be entirely put don’t penetrate the soil like those of down to the very low proportion of most other plants.

New Zealand Garden Journal, 2015, Vol. 18(2) 7 I finally move to a sting in the tail of organic deposits more than 8 m deep, with 800,000 years of Azolla blooms the tale about Azolla. Right now we composed almost entirely of Azolla and a 4,000,000 km² basin to cover, are in a geological period where the fossils, extended along the 1800 km easily enough carbon could have actions of a single organism, human ridge. The finding created a storm of been sequestered by Azolla alone to beings, are driving the world’s climate interest amongst geologists. Extant account for the observed 80% drop in into a new state. Global warming Azolla can only tolerate slight salinity, CO2 concentration in the atmosphere. is real, and it has been brought so why was there a huge production Of course other organisms and other about by us humans releasing fossil chain of Azolla plants in the middle processes were involved, but Azolla carbon into the atmosphere. For this, of the Arctic Ocean, and why were is fingered as the main driver, with geologists have coined the word they there precisely when the Earth this period of the Earth’s history “Anthropocene” to describe what is experienced one of the most dramatic now being called the “Azolla Event”. shaping up as a new geological era. climatic changes in its history? Was After that time the atmospheric there a connection between the two CO concentration continued to It is an irony that this warming has 2 events? fall slowly over the next 49 million recently given us evidence of another years due to geological processes event where the global climate has Many geologists think the answer that sequestered even more carbon been changed by the actions of a is “yes”, drawing the following dioxide. Present values are just single organism. Here it is the onset picture. About 50 million years ago, above 400 ppm, having risen from of the Paleocene ice age, and the the Arctic Ocean was largely land- pre-industrial values in the mid-18th organism is our humble Azolla. In locked and centred on the North Century of about 280 ppm. 2004, the reduced ice cover in the Pole, as it is today, but with much Arctic allowed the Arctic Coring warmer temperatures than those So there it is. The humble little Expedition drillship Vidar Viking, seen today. Its only significant marine Azolla, almost ignored when we talk supported by the Swedish and connection was through a long narrow about our native flora, merits the title Russian icebreakers Oden and seaway called the Turgay Strait, “superorganism” given to it by some Sovetskyi Soyuz, to sail close to the which extended southwards across of those describing these dramatic North Pole and drill deep boreholes western Siberia to the equatorial events. Just Google “Azolla Event” to in sediments of the Lomonosov Tethys Ocean. Then, 49 million get a fuller account of the story. And Ridge beneath the Arctic Ocean years ago, the shallow Turgay Strait show some respect the next time you seabed. The cores it recovered were became blocked, and the Arctic pass a red-surfaced pond. sediments recording events stretching Ocean was isolated from the other References back to the age of the dinosaurs oceans, becoming stratified with little Bieleski, R.L. and Ferguson, 80 million years ago when global vertical mixing of its water, so that I.B. (1983). Physiology and temperatures were much warmer its bottom waters became anoxic metabolism of phosphate and than those of today, and temperate (deprived of oxygen). Rainfall was its compounds. In Encyclopedia conditions extended right out to the high: rivers discharged large volumes of Plant Physiology NS 15A: earth’s poles. We know that this of freshwater, creating layers of Inorganic Plant Nutrition (eds: greenhouse climate ended abruptly surface freshwater extending out Läuchli, A.A. and Bieleski, R.L.), when atmospheric levels of CO into the ocean (similar freshwater 2 pp. 422–449. Springer-Verlag, suddenly fell from 3500 ppm in the layers spread out from the Amazon Berlin. early Eocene to 650 ppm during a River today), explaining the ability of Bieleski, R.L. and Läuchli, A.A. brief period 49 to 47 million years ago, Azolla to grow there. As the floating (1992). Phosphate uptake, efflux resulting in a sudden temperature fall Azolla mats became waterlogged and deficiency in the water fern, in the Earth’s climate into an ice age or as storms fragmented them, Azolla. Plant, Cell & Environment with large icecaps at each pole, still the plants died and sank to the 15: 665–673. partly with us today. And those ACEX anoxic sea floor where there was cores turned up a remarkable finding. no decay and the carbon became Lumpkin, T.A. and Plucknett, D.L. Sediments in that critical period, 49 fossilised and removed from (1980). Azolla: botany, physiology million years ago, revealed that thick circulation. Calculations show that and use as a green manure. Economic Botany 34: 111–153.

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