Pinus patula in South Africa: Effect of seed-borne mycoflora on germination, their control and first report of 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.

• –

•Black-yeast fungi

•Anamorph - Hormonema dematioides

•CSNN – Fir and Abies spp.

•Die–back of pine

• Blue stain (only dead timber)

Background - continued Reports of 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

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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

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