Forest Health Dynamics Laboratory

Brown-spot Needle Blight is Emerging in Alabama:

A Serious Threat to Loblolly Pine Plantations

Datta, Debit., Enebak, S.A., Coleman, J.J. and Eckhardt, L.G.

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Background
❑ Globally, increased tree mortality has been observed due to greater frequency and
severity of biotic attack and climatic stressors

❑ Potential increases in tree mortality associated with climate-induced physiological

stress and interactions with other climate-mediated processes such as disease and
insect/pest outbreaks

❑ Successive needle defoliation by pathogens and environmental stress can limit the
tree’s ability to take up water and nutrients, which can affect forest productivity and
carbon stocks

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Motivation of the Study

❑ Loblolly pine is a leading commercial timber
species native to the southeastern US

❑ Naturally, it grows in 15 southeastern states

mostly in combination with shortleaf pine

❑ An emerging problem of needle defoliation Fig. 1 Natural range of loblolly pine

has been encountered over the past three years

❑ Symptoms are typically ranging from needle

discoloration, premature defoliation, thin
crown followed by tree mortality

Fig. 2 Map of loblolly pine foliar damage

❑ Assuming that it can be caused by a single or
observed in stands across the southeastern US
combination of several fungal pathogen (s)
School of Forestry and Wildlife Sciences, Auburn University
Forest Health Dynamics Laboratory

Materials and Methods

Fresh
Needles
Direct Examination and/or
Microscopic Examination

Direct Incubated Needles

Direct Plated
Identification
Surface Sterilization
(10% Bleach) Surface Sterilization (10%
Bleach & 70% Ethanol)
Incubated with Filter
paper, 500 ul Water) Rinse Prior to Plating
(Distilled H2O)
Plated onto Isolation
Media
Incubation (20-25oC
for 5 days to 2 Weeks)
Transfer to Morphological
Identification Media Identification
Fig. 3 Cultural methods of fungi isolation and identification
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Forest Health Dynamics Laboratory

Cont…
Growth media utilized for recovering fungi from needles

1. CMA-PARP (Phytophthora selective)

2. V8-Agar (Phytophthora selective)

3. Carrot Agar (Phytophthora selective)

4. DM+S, DSM+S (Dothistroma selective)

5. APDA & ¼ PDA (Lophodermium & other fungi)

6. Fresh PNA & Dried PNA (Lecanosicta selective & some other fungi)

7. 2% MEA (Diplodia selective & other fungi)

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Needle Sample Processing

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Cont…
Dried Direct
Mycelium Needles

DNA
extraction

PCR (Universal/Species-
specific primers)

Gel Electrophoresis
(~500 base pairs)

PCR Purification
(E.Z.N.A Kit)

Genome Sequencing
(The Laragen Inc.,)

Identification
Confirmation

Fig. 4 Molecular methods of fungi identification

School of Forestry and Wildlife Sciences, Auburn University
Forest Health Dynamics Laboratory

Molecular Analysis

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Phylogenetic Analysis
❑ Purified PCR products were sent to the Laragen Inc., Biotechnology for sequencing

❑ Raw sequences were edited and adjusted using Bioedit software and deposited in
GenBank and BankIT

❑ Phylogenetic and molecular analyses were done by MEGA version 4.0 software

❑ Alignment were completed using ClustalW

❑ Trees were constructed using Neighbor Joining (NJ) and Maximum Likelihood
(ML) methods

❑ 1000 bootstrap replications were performed for NJ analysis

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Loblolly Pine Health Monitoring

❑ Tree health rating was done by visual inspection to determine disease severity as the
proportion of the crown affected;

Less than a third of the crown affected (>1/3)

One-third to two-thirds of the crown affected (1/3 to 2/3)
More than two-thirds of the crown affected (<2/3)

❑ The location of the crown damage was also recorded:

Bottom, middle, or top of the crown

❑ Data were analyzed using MS Excel 2010. Response variable was either “chlorosis
rating per tree” or “defoliation per tree”

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Tree Crown Rating

A B C

Fig. 5 Crown severity rating such as (A) One-third of crown infected (B) One-third to two-third of
crown defoliated/infected & (C) More than two-third to full crown defoliated/infected
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Forest Health Dynamics Laboratory

Results
Region of occurrence

Washington, Colbert, Elmore, Madison,

Crenshaw (AL)
(17 infected stands)

Symptoms

Yellow to brown spots, premature

defoliation, thin crown

Pathogen (s) recovered

Brown-spot needle blight, Lecanosticta

acicola
Rhizosphora needle cast, Rhizosphora
kalkhoffii
Neopestalotiopsis clavispora
Endophyte
Epicoccum nigrum
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Forest Health Dynamics Laboratory

Cont…
Recent Region of occurrence

Wilcox, West Crenshaw County (AL)

Symptoms – Yellow banding, defoliation &

dying trees

Pathogen - Brown-spot fungi?

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Cont…
Region of occurrence

Upson (GA)
Hampton (SC)
(3 infected stands)

Symptoms

Brown needles at the tip, death of the

terminal buds, needle defoliation

Pathogen (s) recovered

Diplodia tip blight, Diplodia sapinea

Diplodia seriata
Endophyte

Trichoderma caerulescens
Hormonema macrosporum

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Forest Health Dynamics Laboratory

Cont…
Region of occurrence

Bibb (AL)
Ware & Camden (GA)
(3 infected stands)

Symptoms

Dead and dying needles, football shaped

fruiting bodies, needle defoliation

Pathogen (s) recovered

Lophodermium needle cast, Lophodermium

spp.

Sydowia polyspora
Endophyte
Hendersonia pinicola
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Forest Health Dynamics Laboratory

Cont…
Region of occurrence

Macon & Colbert (AL)

Nachitoches (LA)
(4 infected stands)

Symptoms

White aecia, browning needles,

defoliation

Pathogen (s) recovered

Pine needle rust, Coleosporium spp.

Brown-spot needle blight, L. acicola

Epicoccum nigrum
Endophyte
Fungal endophyte

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Fungal Pathogens
A

Fig. 6 Disease symptoms and reproductive structures of (A) L. acicola; (B) Coleosporium sp.;
(C) Lophodermium sp. & (D) D. sapinea on loblolly pine
School of Forestry and Wildlife Sciences, Auburn University
Forest Health Dynamics Laboratory

L. acicola Phylogenetic Tree

L. acicola
population that
affecting Pinus
radiata stands in
Spain

L. acicola population that

affecting Pinus radiata stands
in France

L. acicola
population found in
the northeastern
USA affecting
Pinus strobus
stands across the
New England
states

Fig. 7 Maximum likelihood (ML) tree representing L. acicola and three lineages of L. acicola
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Forest Health Dynamics Laboratory

Cont…
❑ Total 904 fungal cultures were recovered from unhealthy loblolly pine needles

❑ They are divided into 58 major groups based on their colony morphology such as colony
shape, size, color, surface texture, height and marginal growth etc.

❑ To date, 28 species of fungi representing 17 families were identified as pathogens,

endophytes and saprophytes followed by morphological and molecular data

❑ Among them, 12 species of fungi represent either pathogens or weak parasites

❑ Conversely, 8 species of fungi represent endophytes, and 8 species represents saprophytes

❑ No sexual states were observed during the investigation of fungal states under microscope

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Forest Health Dynamics Laboratory

Needle Fungi
Pathogen
Endophyte

Saprophyte Saprophyte
24%

Pathogen
52%

Endophyte
24%

Fig. 8 NJ tree representing pathogenic, endophytic & saprophytic fungi found in the needles
School of Forestry and Wildlife Sciences, Auburn University
Forest Health Dynamics Laboratory

Frequency of Fungal Pathogens

60%
50% LA & RK
45% 50% & CS
LP & CS
Pathogen prevalence

40%

Disease Incidence
35% 40% LA & CS
30% LA & RK
25% 30%
Diplodia
20%
sapinea
15% 20% Coleospo
10% rium sp.
R.
5% 10% kalkhoffii
0% Lophoder
0% mium sp.
Individual Two species Three
species Species

Fig. 9 Pathogen prevalence of each species including L. acicola, Lophodermium

sp., Coleosporium sp., D. sapinae and all species combinations

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Forest Health Dynamics Laboratory

Needle Pathogen Distribution Map

Fig. 11 Map of loblolly pine foliar damage observed in stands across the southeastern US

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Loblolly Pine Health Monitoring

P < 0.0001 Year 2019 Year 2019
Chlorosis Year 2020
P < 0.003 Defoliation Year 2020
Proportion of crown affected

0.80 0.80

Proportion of crown affected

0.70 0.70

0.60 0.60

0.50 0.50

0.40 0.40

0.30 0.30

0.20 0.20
0.10 0.10
0.00 0.00
Stand 1 Stand 2 Stand 3 Stand 4 Stand 5 Stand 6 Stand 7 Stand 1 Stand 2 Stand 3 Stand 4 Stand 5 Stand 6 Stand 7

Fig. 12 Frequency of chlorosis and defoliation of healthy and unhealthy loblolly pines in Chatom,
AL at summer 2019 and 2020. Bars represent means and standard errors (N = 70 trees)

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Discussion
❑ LPND in the southeastern US was due in larger part to the brown-spot pathogen L. acicola,
in addition to needle cast Lophodermium spp., needle rust Coleosporium sp. and tip blight D.
sapinae

❑ It also could be caused by a combination of pathogens such as L. acicola and Coleosporium

sp., L. acicola and Lophodermium sp., L. acicola and R. kalkhoffii as well as L. acicola,
Coleosporium sp., and R. kalkhoffii

❑ Pathogen (s) were localized in terms of the needle damage respective to the loblolly pine

❑ Affected trees were repeatedly symptomatic (chlorotic and defoliated) every year

❑ The disease was spreading from symptomatic trees to initially healthy trees

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Discussion
Stands infected by the brown-spot pathogen were mostly situated in the moist areas. Stand
prevalence on those sites ranged from 55% to 99%. Site conditions could explain why stands are
experiencing worse conditions on these sites. Moreover, long-term environmental factors such as
increasing temperature around the infected sites might be associated with increase pathogen
pressure to loblolly pines.

Mean Temperature (oC)

19

18.5

18

17.5

17

16.5

16

15.5
1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017

Fig. 14 Mean temperature around 27 infected stands in the southeastern US from 1980-2017

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Current Activities
❑ The study is investigating the pattern of the disease and nature of spreading across states,
locality, and/or region

❑ The study has expanded field-based sampling across the southeastern US as well as get more
samples from landowners, forest managers, and forest health state cooperators

❑ The study is working on constructing a more detailed needle pathogen distribution map in
the southeastern US

❑ Foliar nutrient contents and total phenolics response to brown-spot needle blight infection
are under investigation

❑ The research continues to associate the environmental factors which may drive the
emergence and spread of LPND

❑ The study is under way analyzing habitat suitability of needle pathogens across the
southeastern US

School of Forestry and Wildlife Sciences, Auburn University

Forest Health Dynamics Laboratory

Acknowledgements
Committee members Lab managers Graduate Students
Dr. Lori Eckhardt Tina Ciaramitaro Sylvester Menanyih
Dr. Scott Enebak Angelica Baker John Mensah
Dr. Jeffrey Coleman Luis Mendez Jessica Ahl

Needle Shooters
Dr. Lori Eckhardt
Kris Bradley
D.R. Stallworth
John Gunter
Forest Health Nathan Baker
State Cooperative Micah Walker
Forrest Fay

School of Forestry and Wildlife Sciences, Auburn University