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Growing Willows in Treated Wastewater as Healthy Forage for Animals

Hussein Maklada, Yan Landau, Efraim Levinson, Amnon Schwartz

In the ancient Jewish texts it is considered to lack both taste and smell, but it seems that some find the willow to be both tasty and nutritious, and if this is not enough, it fulfils a central role in an innovative Israeli-Jordanian study that focuses on dealing with the treated wastewater flowing in open landscapes. And how is Ramat Hanadiv connected to all this?

ערבה

The willow belongs to the Salicaceae family. It is difficult to identify willow species according to their morphology alone due to the morphological similarity among the different species; to date, 350–500 species have been identified around the world. According to the literature, there are four species in Israel, from the Golan to the Dead Sea: brook willow (Salix acmophylla), white willow (S. alba), S. pseudosafsaf and almond willow (S. triandra). In 2017, scientists completed the sequencing of the willow genomes. The willow has attracted extensive research attention around the world, as it is grown as a renewable source of perennial biomass with multiple harvests in the USA, Canada, Sweden and Britain, often involving irrigation with recycled water during summer. Furthermore, the willow tree contains a stock of secondary metabolites in its canopy and bark – phenolic glycosides from the salicylate group. More than 20 such substances have been identified to date; the most

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common are salicin, salicortin, salidroside, helicon and tremulacin. Some of these metabolites have anti-inflammatory, antioxidant and analgesic properties.

There is great variation among species in the composition of the secondary metabolites, which vary among seasons and among the parts of the plant. This variation has not been examined in the willow species growing in the Land of Israel (an area covering Israel and Jordan).

The willow is a deciduous tree; budding begins in early March and the vegetative growth continues until late November, as a function of the weather and the onset of the rainy season. Willows can be propagated from cuttings; the cuttings have a rapid rooting rate and rapid growth. The tree grows successfully on marginal soils with relatively poor aeration. The rate of

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evapotranspiration from the tree is high; therefore, the willow requires high water availability throughout the growing season, and its distribution is limited to the banks of perennial rivers and proximity to springs.

In different places around the world the willow is used as green fodder for grazing animals during the summer, when the herbaceous species are dry and/or have low nutritional value.

In recent years there has been a growing interest in fodder that combines high nutritional values with unique metabolites that may improve the health and well-being of the animals (neutraceuticals). The advantage of the willow as a potential source of fodder in Israel stems from a number of traits: perenniality – it does not need to be replanted each year; a long green season – nine

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months of the year; tolerance of unaerated, saturated soils; rapid vegetative growth from cuttings; and high accumulation of biomass over a number of harvests. Similarly, it tolerates high-density planting and is able to regenerate from its stem after a large number of harvests.

Research hypotheses:

  1. There are differences in the composition and content of unique metabolites among populations of willow
  2. There are populations of willow in Israel that can be grown with non-industrial treated wastewater
  3. Willow trees can be used as fodder for animals, and can improve their well-being by providing relief from inflammation

Research objectives:

  1. Genetic (DNA) and chemical (unique metabolites) characterisation of willow populations in the Land of Israel
  2. Studying the effect of irrigation with recycled water on tree productivity, unique metabolites and nutritional value
  3. Examining the contribution of the willow to the health of the animals consuming it

Research methods, procedures and results:

To genetically characterise the chosen trees, 17 leaf samples were collected for DNA extraction from trees growing in northern (Golan, Valleys, Carmel), central (Judean Mountains) and southern (Northern Negev) Israel; 17 samples were also taken from northern and central Jordan. Genotyping by sequencing and principal components analysis demonstrated the existence of three populations of willow in the Land of Israel. Not one of them belonged to S. alba; therefore, it was decided to treat the sample as a ‘complex of populations’ belonging to S. acmophylla. Moreover, characterisation of the unique metabolites in this sample using targeted and untargeted, advanced HPLC-MS-TOF identified three different clusters representing three different chemotypes of S. acmophylla. A complete match was found between the genotypes and the chemotypes of the willow trees in the sample. A relationship was also found between the population genotype/chemotype and its sensitivity to wheat rust; among the three clusters, two were resistant and one was sensitive to wheat rust.

A comparative experiment to examine the effect of water quality on indices of growth, unique metabolites and nutritional value took place at Ramat Hanadiv on the southern tip of Mt. Carmel.

Trees representing ten species of the genus Salix were compared, with five replicates, under an irrigation regime with either fresh water or recycled wastewater (secondary treatment, non-industrial wastewater), for two years (4 continuous seasons). All populations of willow grew well under irrigation with secondary treated wastewater, even better than when irrigated with fresh water. We found an effect of season and water quality on the willows’ productivity traits, nutritional value and content of some of the secondary metabolites. Irrigation with treated wastewater increased biomass production, and raised the shoot protein content and the leaf salicin content. Leaves and stems were intensively consumed by the goats in the experiment. In the first experiment, with 24 Baladi goats, leaf and stem consumption had no negative effect on milk production and reduced the number of somatic cells in the milk, as an estimate of intra-mammary infection and mammary involution at the end of lactation. The concentration of liver enzymes indicated that fattening with willow was very safe. The mineral content of the consumed fodder did not exceed the accepted range. In the second experiment, which was performed on 48 Alpine crossbred goats in late pregnancy, willow consumption constituted 25% of their food portion. The food portion contained 75 g salicin per kg dry matter. The proportion of CD8+ T-cells, common under conditions of stress and inflammation – was identical between treatments at the beginning of the experiment and increased greatly in the blood of all the control goats. In the willow group there was a small increase, but only in the goats infected with intra-mammary infection vectors. Therefore, consumption of willow foliage probably led to changes in the distribution of immune cells in the goats’ blood. It is possible that the components in willow have an immuno-regulatory effect, regardless of intra-mammary infection.

In conclusion, most of the willow trees growing in nature in the Land of Israel belong to the population complex of S. acmophylla, in which three different genotype/chemotypes were identified. Although it was previously though that S. alba was the dominant species in northern Israel, 33 of the 34 samples from the entire Land of Israel belonged to other species. Willow productivity was higher under an irrigation regime with treated wastewater. Willow populations with rapid growth and high content of secondary compounds were identified. The willow is a fodder source with good nutritional traits (high protein, ca. 12% on average, and moderate digestibility, ca. 50%), and properties for relieving stress and inflammation at the end of lactation in goats.

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