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Pinus Patula in South Africa: Effect of Seed-Borne Mycoflora on Germination, Their Control and First Report of Sydowia Polyspora

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Pinus Patula in South Africa: Effect of Seed-Borne Mycoflora on Germination, Their Control and First Report of Sydowia Polyspora Pinus patula in South Africa: Effect of seed-borne mycoflora on germination, their control and first report of Sydowia polyspora Prof. Terry Aveling (Seed Science, UP) Renaan Thompson (PhD Plant Pathology) Dr Mervyn Beukes (Biochemistry, UP) Dr Guro Brodel (Nibio, Norway) 1 Background • South African pine plantations - 3.4% to South Africa’s GDP, covered 607 815 ha during 2015/2016 • Forestry sector in S. A. employs around 165 900 workers directly • P. patula least tolerant to disease of 3 main species grown in South Africa - P. elliottii (most), P. taeda (moderately) • Pinus patula is native to Mexico • Evergreen conifer. Long (25 cm), light green, drooping needles MAP LEGEND optimum (green) mortality (black) too dry regions (yellow) 2 Schulze and Maharaj, 2007 Background - continued Sydowia polyspora (Bref. & Tavel) E. Müll., (1953) •Described on Pinus sp. •Ascomycota – Dothideales •Black-yeast fungi •Anamorph - Hormonema dematioides •CSNN – Fir and Abies spp. •Die–back of pine • Blue stain fungus (only dead timber) Background - continued Reports of Sydowia polyspora and it’s synomorph Hormonema dematioides 2010 2014 In this publication the fungus Kabatina abietis was renamed Sydowia polyspora and was proven to be the causal agent of 4 the disease CSNN Background - continued Reports of Sydowia polyspora and it’s synomorph Hormonema dematioides 2014 2016 S. polyspora dominated P. radiata symptomatic and asymptomatic needles 5 Background - continued Reports of Sydowia polyspora and it’s synomorph Hormonema dematioides 2014 2018 The pathogenicity of the fungus is proven and also its association with the pine bark beetle – Tomicus piniperda 6 Background - continued Reports of Sydowia polyspora and it’s synomorph Hormonema dematioides 2014 ???? 7 The aim of this study was to screen Pinus patula seed for the presence of seed-borne mycoflora Materials and Methods Pine seed treatment and standard germination test • Germination tests done according to ISTA Treatment 2) Stratification: 1) Bubbling of the seed as 1 4ºC for 1, 3, 5, 14 and 21d done by some forestry seed companies 3)Treatment with NaOCl and H2O2; Different time intervals and concs 2 Source seed 3 Germination test 7 seed lots 7 and 21 d 9 Materials and Methods - continued Isolation and identification of seed-borne fungi Source seed and plant Surface disinfestation Plating out - 43 seed lots Morphological and Molecular ID Storage Incubation Evaluation Materials and Methods - continued Sequencing DNA extraction Quantification PCR with ITS, β- tubulin and RPB2 BLAST - Genbank Gel electrophoresis Sequencing Alcohol precipitation Sequencing PCR Purification of DNA 11 Materials and Methods - continued Pathogenicity tests Spore suspension 107/ml Wounded and unwounded Detached needles: Seedling symptom Pure cultures Stab inoculation with test: Needles gently 5 Isolates sterile toothpicks rubbed together. Control Needles and fungus Seedlings covered Incubation 100% RH for with polythene bags 7 d for 5 d (greenhouse) Re-isolation Evaluated after 14 d Evaluation (DI/DS) Results Seed germination results of Pinus patula seed lots Abnormal seedlings (%) Normal Non- Seed lot seedlings germinated Pinus species number (%) (%) Deformed Diseased P. patula PP 64261 31 27 0 42 P. patula PP 69252 45 32 1 22 P. patula PP 67164 38 42 0 20 P. patula PP 10360 35 20 0 45 P. patula PMP 16331 42 38 1 19 P. patula PP 66919 44 24 0 32 P. patula PP 69130 40 29 0 31 Results - continued • Bubbling and stratification reduced % germination • NaOCl at concentration of 0.5% (for 1 and 3 min) and 1% (1 min) increased % germination by 3, 5 and 6%, resp. • H2O2 at concentration of 1% (for 1 and 3 min) and 1.5% (2 min) increased % germination by 5, 5 and 7%, resp. 14 Results - continued Amount of fungal isolates from the different seed lots of Pinus patula Fungal species Total fungal Pinus species code isolates 1/2 PDA MGA PP 64261 PP 64261 (1-59) 59 42 17 PP 69252 PP 69252 (1-103) 103 79 4 PP 67164 PP 67164 (1-45) 45 34 22 PP 10360 PP 10360 (1-49) 49 36 13 PMP 16331 PMP 16331 (1-61) 61 49 12 PP 66919 PP 66919 (1-81) 81 67 14 PP 69130 PP 69130 (1-56) 56 46 10 Total 451 341 110 15 Results - continued • Sydowia polyspora was isolated from 4 seed lots of Pinus patula • Morphologically identified using compound microscopy: 1–2 conidiogenous loci per cell in S. polyspora with melanized hyphae/ chlamydospores • BLAST’s with all three gene regions resulted in 99, 98 and 89% identification (PPRI 25873) 16 Results - continued • DI: ≈100% - wounded and unwounded • DS: 15 – 25% - in all inoculated seedlings • Characteristic symptoms: yellowing and browning from tip of some leaves and from point of the wound as well as banding. First chlorotic then necrotic • Uninoculated needles and seedlings – no symptoms 17 Discussion • Fungus isolated from pine seed in Norway (2010) • Pathogenicity proven on other Pinus species in Italy, Spain, Tasmania and China • Sydowia polyspora in South Africa on P. patula seed • Pathogenicity proven on P. patula detached needles and one year old seedlings • Presence of fungus may negatively affect the industry; movement by means of seed; can be a quarantine pathogen in certain countries; can affect export/import of pine seed 18 19 .
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