Fig 1.
An adaptation of the proposed karrikin signal transduction mechanism in plants.
(A) Exposure of seeds to exogenous karrikins is proposed to trigger a molecular response (B) whereby the karrikin binds an α/ß-hydrolase called KAI2 that induces a conformational change in protein shape, thus allowing KAI2 to interact with MAX2, an F-box subunit of an SCF class of E3 ubiquitin-protein ligase complex. SCFMAX2 then degrades growth repressing proteins, increasing a seed’s sensitivity to light and stimulating plant developmental processes (C) such as enhanced seed germination, seedling photomorphogenesis, leaf morphogenesis and possibly stress resilience [4,5].
Fig 2.
A schematic diagram of the systematic concept used for biochar manufacture.
Biochars were made in sizeable quantities by three commercially operating pyrolysis technologies that included a slow pyrolyser designed for high through-put requirements (Technology A, Tech A); a slow pyrolyser for medium volume needs (Tech B) and a mobile fast pyrolyser for on-farm processing (Tech C). Six study biochars were prepared; a common green waste feedstock was made into three biochars by the commercial units, a woodchip and paper mill waste feedstock were made into biochar by a batch reactor version of Tech A (20 L capacity) and sugarcane trash was made into biochar by the commercial Tech C unit to simulate an on-farm feedstock. Additionally, a liquid bi-product was produced by Tech B.
Table 1.
The conditions that prevailed during biochar manufacture.
Biochars were made in sizeable quantities by three commercially operating pyrolysis technologies A, B or C or a batch reactor version of Tech A. Inputs were a common green waste feedstock made into biochar by all commercial technologies. Additionally, biochars were made from a sugarcane trash, paper mill waste or woodchip feedstock to expand the study scope.
Fig 3.
The yield of karrikinolide (KAR1) isolated from study biochars as related to pyrolysis conditions and input feedstocks.
Biochars were made by Technologies A, B or C in commercial units from a green waste or sugarcane trash feedstock or by a 20 L sized batch reactor using a paper mill waste or woodchip feedstock. KAR1 yield is represented as mean ± SEM in each study biochar (ng KAR1 per 100 g of biochar, n = 3 replicates). Analysis of variance compared KAR1 abundance within different biochars (F5,11 = 79.51, P < 0.001; data log10 transformed prior to analysis); values with different letters are significantly different from one another (Tukey test, α = 0.05).
Table 2.
Selected physiochemical properties of study biochars.
Biochars were made by Technologies A, B or C in commercial units from a green waste or sugarcane trash feedstock or by a 20 L sized batch reactor using a paper mill waste or woodchip feedstock.
Fig 4.
Tomato seedling size in preliminary glasshouse biochar trials at two weeks after germination.
Plants were grown in a peat mixture without biochar (control) or with peat replaced at 3, 10 or 30% by either a green waste biochar high in KAR1 or a sugarcane biochar low in KAR1. All mixtures contained fertiliser to neutralise potential nutritional aspects of biochar [19]. Values represent means ± SEM (n = 5 biological replicates). Analysis of variance and means tested the effect of biochar type and rate of application on (a) shoot length; (b) hypocotyl length; (c) length of the largest fully open leaf; (d) average shoot weight per seedling; stars indicate means that are significantly different to the control (lsd, α = 0.05).
Fig 5.
The seed germination response at 14 d after sowing of two model species that require karrikinolide (KAR1) to break dormancy, Solanum orbiculatum (SO, a-d) and Brassica tournefortii (BT, e-h), to water, synthetic KAR1, biochar extracts or a liquid by-product from Tech B. Bars represent mean ± SEM (biological replicates: SO, n = 3; BT, n = 5). The control (a, e) was Milli-Q water and treatment extracts were prepared from (b, f) karrikin-abundant biochars or (c, g) low-karrikin biochars and (d, h) a liquid by-product of Tech B pyrolysis. Biochar extracts were diluted to concentrations equivalent to 50, 10, 2.5 and 1% biochar application rates and made from green waste by Technologies A, B or C, sugarcane trash by Technology C or woodchips by Technology A. The liquid by-product from Tech B was diluted to 1/10 (one part liquid to nine parts Milli-Q water), 1/100, 1/500, 1/1000 and 1/5000 or 1/2000. Seeds germinated on 0.67 μM KAR1 solution (a, e) were not included in analyses (included to confirm seed KAR1 sensitivity and viability). Seed germination across all other treatments was compared using analysis of variance within each species (SO, F25,56 = 53, P < 0.001; BT, F29,115 = 143, P < 0.001); stars indicate means that are significantly greater than the water control within a species (Dunnett test, α = 0.05). Values were arcsine transformed prior to analysis.
Fig 6.
Biochar dosage response curves for tomato and lettuce seedlings at one week after sowing.
Biochar extracts were diluted to concentrations equivalent to 1, 2.5, 10 and 50% application rates (shown on a logarithmic scale in a-d) and made from green waste by Techologies A, B or C, sugarcane trash by Technology C, papermill waste by Technology A or woodchips by Technology A. Analysis of variance compared shoot and root length across treatments for (a, c) tomato and (b, d) lettuce (tomato, F24,95 = 21.28shoot, 18.68root, P < 0.001; lettuce, F24,96 = 46.85shoot, 46.23root, P < 0.001); means above the upper line or below the lower line are significantly different to the control (Dunnett test, α = 0.05).
Fig 7.
The relationship between the plant phenotype and biochar properities, including karrikin content, for tomato and lettuce seedlings at one week after sowing using multivariate analysis.
Multivariate analyses, plotted on principle components 1 (PC1, x-axis) and 2, indicate an association between biochar KAR1 content and shoot length for (a) tomato grown on 10% biochar extracts (PC1 accounted for 72.7% and PC2 for 16.9% of the variation) and (b) lettuce on 50% biochar extracts (PC1 = 73.1% and PC2 = 13.8%). At other biochar doses there were no associations between plant phenotype and KAR1 content of biochar. Plant phenotype: shoot length, root length (lettuce and tomato), secondary root number (lettuce). Biochar properties: KAR1 content, nitrogen, potassium, sodium, EC, pH and heavy metals of concern: chromium, lead, arsenic (tomato and lettuce) and nickel (lettuce). Modelled heavy metals exceeded minimum environmental standards in green waste biochars (Table 2) and/or were at levels that elicit phytotoxicity for the study species [39]. Phosphorus did not change in leaf tissues between treatments (S2 Fig) and substantially reduced the variance explained by the first two components, hence was not modelled.