EFFECT OF BIOCHAR ON PLANT GROWTH AND DEVELOPMENT IN STRAWBERRY, PEACH, APPLE AND NECTARINE

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1 EFFECT OF BIOCHAR ON PLANT GROWTH AND DEVELOPMENT IN STRAWBERRY, PEACH, APPLE AND NECTARINE Lidia Sas Paszt, Mateusz Frąc, Edyta Derkowska, Sławomir Głuszek, Paweł Trzciński Research Institute of Horticulture UNDERSTANDING BIOCHAR MECHANISMS FOR PRACTICAL IMPLEMENTATION SEPTEMBER 2015 HOCHSCHULE GEISENHEIM UNIVERSITY

2 RHIZOSPHERE LABORATORY The role of roots & the rhizosphere in the growth & yielding of fruit plants. Development of sustainable methods of cultivation & fertilization of fruit plants for the production of high quality fruit and to increase the natural fertility of the soil using PGPR rhizobacteria, AMF fungi and other components of the soil biosphere.

3 INTRODUCTION Biochar (47 t/ha, 5 kg/tree) or a mixture of biochar and compost (47 t/ha:10 t/ha), applied in an orchard replanted with 1 yo apple cv. Fuji on M26 increased trunk diameter by 5% and 10%, respectively, compared to the control [Ayles et al. 2014]. Application of biochar (50 t/ha) has positive effect on growth and nutrition of M26 apple rootstocks, compared to control [Street et al. 2014]. A hypha of a mycorrhizal fungus on the surface of an apple-tree root.

4 Application of biochar from coniferous trees significantly increased growth of peach trees grafted on rootstocks susceptible to replant disease. Biochar may alleviate replant disease in peach trees [Atucha and Litus 2015]. Bacteria in root cells of a peach tree. Hyphae of a mycorrhizal fungus on the surface of a root of a peach tree treated with biochar.

5 In strawberry, biochar induces a systemic response to foliar fungal pathogens such as Botrytis cinerea, Colletotrichum acutatum or Podosphaera aphanis. Plants growing in a substrate with a 3% addition of biochar exhibited the lowest disease severity compared to control plants [Meller Harel et al. 2012]. Application of biochar to a fertile soil reduced the number of deformed and damaged fruits, in comparison with control [Jael et al. 2012].

6 BIOCHAR USED IN THE EXPERIMENTS Derived from waste biomass wood chips of conifers Biochar particle size up to 50 mm Produced by fast pyrolysis 280 C Organic matter content 80% Organic carbon content 20% Moisture content at the time of application 15% Mineral content in the fresh mass of sample ph (in H 2 O) 8.4 Nitrogen (N) 0.64% Phosphorus (P 2 O 5 ) 0.28% Potassium (K 2 O) 0.75% Calcium (CaO) 2.52% Magnesium (MgO) 0.16% MAGNIFIED VIEW OF BIOCHAR

7 GREENHOUSE EXPERIMENT WITH STRAWBERRY The strawberry plants were planted in sand or in soil mixed with 2 doses of biochar. All the plants were equally fertilized with standard NPK fertilizer. Two levels of biochar (1 and 2.5 g/plant) were assessed in terms of their effect on plant growth and development, as compared with the control. Control Biochar level 1 Biochar level 2 Control Biochar level 1 Biochar level 2

8 Biochar used in the cultivation of potted strawberry plants increased the number of leaves compared with the control.

9 Application of 2.5 g of biochar markedly increased the weight of the root system of strawberry plants growing in the sand (by 20%), as well as those growing in the soil (by 34%). The use of 2.5 g of biochar increased the formation of strawberry crowns.

10 INFLUENCE OF BIOPREPARATIONS ON THE COLONIZATION OF STRAWBERRY ROOTS BY AMF TREATMENT F% (mycorrhizal frequency) Number of spores Control b 58 c Control NPK 7.78 a 35 a Manure c 72 d Mycosat f 83 e Humus UP e 47 b Vinassa c 59 c Florovit Natura c 81 e Bacterial consortium d 50 b Mycorrhiza + bacteria d 62 c Spores in the roots of ELSANTA strawberry plants (July 2013). Biochar + bacteria e 83e The applied bioproducts increased mycorrhizal frequency in the roots & the number of spores of AMF in the rhizosphere. Vesicles in the roots of ELKAT strawberry plants (July 2013) Derkowska E., Sas Paszt L., Harbuzov A., Sumorok B., Bogumił A. Root growth, mycorrhizal frequency and soil microorganisms in strawberry as affected by biopreparations. Journal of Plant Nutrition (in press).

11 MYCORRHIZAL STRUCTURES IN THE ROOTS OF ELSANTA STRAWBERRY PLANTS A B C A Mycorrhizal mycelium and spore in the roots - Micosat (mag. 10 x 40) B Vesicles in the roots Biochar + rhizosphere bacteria (mag. 10 x 40) C Root fragment with mycelium, vesicles, and arbuscules inside it - Humus UP (mag. 10x10) Application of biochar with rhizospheric bacteria increased the formation of mycorrhizal structures in strawberry roots.

12 ORCHARD EXPERIMENT WITH APPLE AND PEACH TREES Apple and peach trees were planted in the spring of Biochar from coniferous wood chips (with a moisture content of 15%, Fluid S.A.) was applied at a dose of 1.6 kg per tree in the spring of Biochar was sprinkled on an area of 2 m 2 in the form of a ring and mixed with the top layer of soil. Each combination consisted of 4 replicates, each replicate of 3 trees. Florovit NPK fertilizer: N - 5%, P 2 O 5-3%, K 2 O - 2%, OA - 30%. Control Biochar 1.6 kg/tree Biochar 1.6 kg/tree + Microorganisms - Pseudomonas fluorescens Ps1/2 and Pantoea sp. N52AD) + AMF fungi Biochar 1.6 kg/tree + Florovit NPK (200g/tree) Application of biochar in the orchard

13 PARAMETERS MEASURED in 2015: Number of bacteria and microscopic fungi Soil moisture Characteristics of vegetative growth of above-ground parts Characteristics of root growth Mycorrhizal frequency Total yield, number & size of fruits Soil fungus isolated from soil Hyphae of mycorrhizal fungi on root surface

14 NUMBER OF BACTERIA AND MICROSCOPIC FUNGI IN THE SOIL Treatments COLLECTED FROM UNDER MEREDITH PEACH TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Soil dry weight (%) Total number of bacteria (cfu) in 1 g DW 10 7 Total number of microscopic fungi (cfu) in 1 g DW 10 4 Control 88.5 b 53.5 c 69.3 a Biochar 85.6 ab 19.7 a 74.3 ab Biochar + Microorganisms 84.3 a 37.2 b 95.3 b Biochar + Florovit NPK 86.6 ab 33.1 b 87.0 ab Application of biochar alone or in conjunction with microorganisms or Florovit NPK contributed to a reduction in the number of isolated bacteria and to a slight increase in the number of microscopic fungi. Mycorrhizal fungi on the surface of a peach-tree root Penicillium fungus isolated from biochar-treated soil

15 GROWTH CHARACTERISTICS OF MEREDITH PEACH-TREE ROOTS (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Root fresh weight [g/1l soil] Root dry weight [g/1l soil] Control 2.21 a 1.21 a Biochar 3.18 ab 1.68 a Biochar + Microorganisms 5.18 c 2.73 b Biochar + Florovit NPK 3.91 b 1.51 a Application of biochar with beneficial microorganisms significantly increased fresh and dry weight of roots of Meredith peach trees. Mycorrhizal mycelium entangling a peach-tree root

16 GROWTH CHARACTERISTICS OF MEREDITH PEACH-TREE ROOTS Treatment Root length [cm/1l soil] Root surface area [cm 2 /1L soil] Root diameter [mm/1l soil] Root volume [cm 3 /1L soil] Number of root tips [per 1L soil] Control a 72.9 a 0.47 a 0.83 a 2183 a Biochar ab ab 0.42 a 1.08 a 4118 ab Biochar + Microorganisms Biochar + Florovit NPK (ORCHARD EXPERIMENT, DĄBROWICE, 2015) ab 91.7 a 0.57 a 1.21 ab 3279 ab b b 0.41 a 1.66 b 5305 b Application of biochar with Florovit had a positive effect on the size of the root system of Meredith peach trees. Mycorrhizal fungi on the surface of a peach-tree root Absence of mycorrhizal structures in the roots of control peach trees

17 MICROSCOPIC STUDY OF THE ROOTS OF APPLE, PEACH AND NECTARINE TREES TREATED WITH BIOCHAR METHODS Comparative morphological assessment of the root system of each species was performed using a Canon PowerShot SX10 camera. Representative samples were treated with hot (60 C) 1% NaOH for measurements of the thickness of roots and vascular bundles with an Olympus SZX 16 stereoscopic microscope and Cell BR program. Examination of differences between objects at the level of tissues and cells was conducted with histological preparations of roots fixed in CrAF (chromic acid, acetic acid, formalin), dehydrated in ethanol and embeded in paraffin, cut with microtome to 15 µm thick cross-sections and stained with safranine and fast green were made. The material was analyzed with a Nikon Eclipse 80i light microscope with polarization and NIS-Elements Br 2.30 program. Vesicles in the roots of strawberry Arbuscules in the roots of strawberry

18 SAMPLES OF PEACH TREE ROOTS GROWING IN BIOCHAR SUBSTRATES Control Biochar Biochar + Florovit NPK The biochar treated plants had a far greater density of the finest, young roots than in the control roots, having the least branched root system.

19 MEASUREMENTS OF THE THICKNESS OF THE ROOTS AND VASCULAR BUNDLES IN PEACH, PERFORMED ON STEREOSCOPIC MICROSCOPE IMAGES vascular bundle Relationship between the thickness of vascular bundles-xylem and the thickness of the roots of peach In peach, there were no differences between the control and biochar treatment in the development of xylem.

20 THE EFFECTS OF BIOCHAR ON STARCH PRESENT IN PEACH ROOT CELLS xylem Starch grains Cross-sections of peach roots in normal and polarized light The roots of peach trees had the cortex cells filled with starch in the combinations with biochar.

21 TRUNK DIAMETER, HEIGHT AND SIZE OF MEREDITH PEACH TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Trunk dia. Tree height Tree size [mm] cm] [m 2 ] Control 40.4 a 255 a 4.7 a Biochar 55.7 c 284 b 5.9 b Biochar + Microorganisms 49.4 b 277 b 5.2 ab Biochar + Florovit NPK 49.0 b 284 b 5.3 ab Biochar at the dose of 1.6 kg/tree had a positive effect on the vegetative growth of Meredith peach trees.

22 NUMBER OF FRUITS, YIELD, AND FRUIT SIZE OF MEREDITH PEACH Treatment (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Number of fruits [per tree] Total fruit yield [kg/tree] Mean fruit weight [g] Control 212 ab 14.1 a 65.6 a Biochar 267 b 18.6 b 70.4 a Biochar + Microorganisms 265 b 18.2 b 68.5 a Biochar + Florovit NPK 157 a 13.1 a 85.5 a A fruiting Meredith peach tree Application of biochar and biochar with microorganisms contributed to a significant increase in the number of fruits and total yield. The largest fruits were obtained from the trees treated with biochar and Florovit NPK, and the smallest from the control trees.

23 MYCORRHIZAL FREQUENCY IN THE ROOTS OF MEREDITH PEACH TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment F% M% m% Observations Control 8.89 ab 0.09 ab 1.0 b Single vesicles Biochar 8.89 Numerous 0.09 ab 0.7 ab ab vesicles Biochar + Numerous 1.11 a 0.01 a 1.0 b Microorganisms vesicles Biochar + Florovit NPK Numerous 0.11 b 1.0 b b vesicles F% - mycorrhizal frequency M% - relative mycorrhizal frequency m% - non relative mycorrhizal frequency Application of biochar + Florovit NPK had a positive effect on mycorrhizal frequency in the roots of Meredith peach trees. AMF spore on a root of a Meredith peach tree treated with biochar and Florovit NPK Vesicles in the roots of Meredith peach trees treated with biochar and Florovit NPK

24 NUMBER OF BACTERIA AND MICROSCOPIC FUNGI IN THE SOIL COLLECTED FROM UNDER ARIWA APPLE TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Soil dry weight (%) Total number of bacteria (cfu) in 1 g DW 10 7 Total number of microscopic fungi (cfu) in 1g DW 10 4 Control 90.4 a 59.7 a 82.9 a Biochar 94.0 ab 59.9 a 68.5 a Biochar + Microorganisms 91.1 a 62.2 a 90.8 a Biochar + Florovit NPK 95.0 b 65.6 a c Application of biochar and Florovit NPK did not affect the number of isolated bacteria, but it increased the total number of microscopic fungi. Root of apple tree treated with NPK fertilizer. Hyphae of soil fungi under microscope

25 GROWTH CHARACTERISTICS OF ARIWA APPLE-TREE ROOTS (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Root fresh weight [g/1l soil] Root dry weight [g/1l soil] Control 1.78 a 0.80 a Biochar 4.16 b 1.73 b Biochar + Microorganisms 5.35 b 2.50 c Biochar + Florovit NPK 3.36 ab 1.45 ab Application of biochar alone or biochar enriched with beneficial microorganisms contributed to a significant increase in fresh and dry weight of the roots of Ariwa apple trees.

26 GROWTH CHARACTERISTICS OF ARIWA APPLE-TREE ROOTS Treatment (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Root length [cm/1l soil] Root surface area [cm 2 /1L soil] Root diameter [mm/1l soil] Root volume [cm 3 /1L soil] Number of root tips [per 1L soil] Control a 65.4 a 0.59 a 0.95 a 1710 a Biochar a 79.6 ab 0.99 b 1.89 bc 1068 a Biochar + Microorganisms a b 0.84 ab 2.29 c 1584 a Biochar + Florovit NPK a 68.6 a 0.79 ab 1.33 ab 1077 a The combined use of beneficial microorganisms and biochar increased the length, surface area and volume of the roots of Ariwa apple trees. A spore of a mycorrhizal fungus Hyphae of a mycorrhizal fungus on the surface of a root of an apple tree treated with biochar.

27 SAMPLES OF APPLE TREE ROOTS TREATED WITH BIOCHAR Control Biochar Biochar +Microorganisms The finest roots of the plants treated with biochar were several times longer and more compacted than in the control.

28 MEASUREMENTS OF THE THICKNESS OF THE ROOTS AND VASCULAR BUNDLES IN APPLE PERFORMED ON THE STEREOSCOPIC MICROSCOPE IMAGES vascular bundle

29 EFFECTS OF BIOCHAR ON CELL STRUCTURE OF APPLE ROOTS cork cortex phloem xylem xylem crystals of calcium compounds CONTROL starch grains BIOCHAR Cross-sections of apple roots under normal and polarized light The roots of apple trees, like those of peach trees, had the cortex cells filled with starch in the combinations with biochar.

30 TRUNK DIAMETER, HEIGHT AND SIZE OF ARIWA APPLE TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Trunk diameter [mm] Tree height [cm] Tree size [m 2 ] Control 30.3 a 204 a 1.94 ab Biochar 33.2 ab 196 a 1.60 a Biochar + Microorganisms 35.1 b 210 a 1.73 a Biochar + Florovit NPK 36.9 b 232 b 2.33 b Application of biochar with Florovit NPK contributed to a significant increase in the vegetative growth of apple trees, e.g. trunk diameter, height and size of trees.

31 MYCORRHIZAL FREQUENCY IN THE ROOTS OF ARIWA APPLE TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment F% M% m% Observations Control 0.0 a 0.0 a 0.0 a No mycorrhizal structures Biochar 1.11 a 0.01 a 1.0 b Single vesicles Biochar + Microorganisms Biochar + Florovit NPK 7.78 b 0.08 b 1.0 b 0.0 a 0.0 a 0.0 a Application of biochar with microorganisms significantly increased the colonization of roots by mycorrhizal fungi. No mycorrhizal structures in the roots of control apple trees. Numerous vesicles in the roots of Ariwa apple trees treated with biochar and beneficial microorganisms Numerous vesicles No mycorrhizal structures F% - mycorrhizal frequency M% - relative mycorrhizal frequency m% - non relative mycorrhizal frequency

32 ORCHARD EXPERIMENT WITH NECTARINE TREES In the spring of 2014, nectarine trees of Prunus persica Flateryna were treated with biochar in doses: Control no biochar Biochar 0.8 kg/tree Biochar 1.6 kg/tree Biochar 2.4 kg/tree Nectarine trees were planted in the spring of Biochar of coniferous wood chips (a moisture content of 15%, Fluid S.A.) was applied in the spring of The biochar was sprinkled on an area of 2 m 2 in the form of a ring and mixed with the top layer of soil. Each combination consisted of 3 replications, and a replication of 3 trees.

33 NUMBER, YIELD, AND SIZE OF NECTARINE FRUITS (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Control Biochar 0.8 kg/tree Biochar 1.6 kg/tree Biochar 2.4 kg/tree Number of fruits [per tree] Total fruit yield [kg/tree] Mean fruit weight [g] 175 a 10.5 ab 60.7 ab 173 a 11.5 b 67.5 b 172 a 10.1 ab 58.9 ab 151 a 8.6 a 56.8 a The highest yield and mean fruit weight was obtained from the trees treated with biochar at a dose of 0.8 kg per tree.

34 NUMBER OF BACTERIA AND MICROSCOPIC FUNGI IN THE SOIL COLLECTED FROM UNDER NECTARINE TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Soil dry weight (%) Total number of bacteria (cfu) in 1 g DW 10 7 Total number of microscopic fungi (cfu) in 1g DW 10 4 Control 88.1 b 11.4 a 38.2 a Biochar 0.8 kg/tree 87.7 ab 13.0 a c * Biochar 1.6 kg/tree 85.7 a 10.0 a 56.4 ab Biochar 2.4 kg/tree 86.6 ab 17.6 b 82.7 b Biochar at a dose of 0.8 kg/tree significantly increased the total number of microscopic fungi. Application of biochar at a dose of 2.4 kg/tree contributed to a significant increase in the total number of bacteria isolated from the soil.

35 MYCORRHIZAL FREQUENCY IN THE ROOTS OF NECTARINE TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment F% M% m% Observations Control 18.9 b 0.19 b 1.0 a Single vesicles Biochar 0.8 kg/tree Biochar 1.6 kg/tree Biochar 2.4 kg/tree 23.3 b 0.23 b 1.0 a Numerous vesicles 14.0 ab 0.14 ab 1.0 a Single vesicles 10.0 a 0.10 a 1.0 a Single vesicles Mycelium of a mycorrhizal fungus in a root of a nectarine tree treated with biochar at 0.8 kg/tree. Application of biochar at 0.8 kg/tree increased mycorrhizal frequency and the number of vesicles observed in the roots of nectarine trees.

36 GROWTH CHARACTERISTICS OF NECTARINE TREE ROOTS (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Root fresh weight [g/1l soil] Root dry weight [g/1l soil] Control 3.33 a 1.65 a Biochar 0.8 kg/tree 3.93 ab 2.06 b Biochar 1.6 kg/tree 2.45 a 1.20 a Biochar 2.4 kg/tree 3.01 a 1.46 a Biochar at a dose of 0.8 kg/tree increased the fresh & dry weight of roots of nectarine trees. Mycorrhizal mycelium in the roots of nectarine trees treated with biochar at 0.8 kg/tree.

37 GROWTH CHARACTERISTICS OF NECTARINE TREE ROOTS (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Treatment Root length [cm/1l soil] Root surface area [cm 2 /1L soil] Root diameter [mm/1l soil] Root volume [cm 3 /1L soil] Number of root tips [szt/1l soil] Control a 66.9 a 0.63 ab 1.06 a 1478 a Biochar 0.8 kg/tree Biochar 1.6 kg/tree Biochar 2.4 kg/tree ab 85.4 b 0.86 b 1.54 b 1923 ab b 80.8 ab 0.46 a 0.91 a 2915 b a 69.1 a 0.63 ab 1.06 a 1680 a Application of biochar at 0.8 kg/tree and 1.6 kg/tree affected favourably the growth characteristics of nectarine tree roots. A spore in a root of a nectarine tree treated with biochar at 0.8 kg/tree

38 SAMPLES OF NECTARINE TREE ROOTS TREATED WITH BIOCHAR Control 0.8 kg/tree 2 t/ha 2.4 kg/tree-6 t/ha At the doses of biochar a marked increase in the number and length of young roots was observed, indicating favourable properties of biochar on root formation.

39 MEASUREMENTS OF THE THICKNESS OF THE ROOTS AND VASCULAR BUNDLES IN NECTARINE, PERFORMED ON STEREOSCOPIC MICROSCOPE IMAGES vascular bundle xylem Control 0.8 kg/tree 1.6 kg/tree 2.4 kg/tree Relationship between the thickness of vascular bundlesxylem and the thickness of the roots of nectarine In the nectarine, the use of the highest dose of biochar had the most favourable effect on the development of xylem. The highest bar represents thicker xylem bundles at the highest dose of biochar.

40 THE EFFECTS OF BIOCHAR ON STARCH PRESENT IN NECTARINE ROOT CELLS xylem Starch grains Cross-sections of nectarine roots in normal and polarized light The roots of nectarine trees, like those of peach and apple trees, had the cortex cells filled with starch in the combinations with biochar.

41 TRUNK DIAMETER, HEIGHT AND SIZE OF NECTARINE TREES (ORCHARD EXPERIMENT, DĄBROWICE, 2015) Combinations Trunk diameter [mm] Tree height [cm] Tree size [m 2 ] Control 52.5 b 265 b 5.0 a Biochar 0.8 kg/tree Biochar 1.6 kg/tree Biochar 2.4 kg/tree 50.6 ab 263 b 4.6 a 46.7 a 252 ab 4.4 a 48.1 ab 248 a 4.3 a Appication of biochar had no effect on trunk diameter, tree height and tree size. Applying biochar

42 CONCLUSIONS Addition of biochar to the soil in 2014 increased the total number of microscopic fungi, mycorrhizal frequency in the roots, and contributed to the increase in the vegetative growth and yielding of the apple, peach and nectarine trees in Application of biochar increased formation of young roots, development of xylem and starch formation in the cells of the roots, but reduced the deposition of corky cells on the surface of roots. Spores of mycorrhizal fungi (AMF) during isolation

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