dattadebitsigmaxi2021

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

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

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
has been encountered over the past three years
 Symptoms are typically ranging from needle
discoloration, premature defoliation, thin
crown followed by tree mortality
 Assuming that it can be caused by a single or
combination of several fungal pathogen (s)
Fig. 1 Natural range of loblolly pine
Fig. 2 Map of loblolly pine foliar damage
observed in stands across the southeastern US

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

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)

Needle Sample Processing

Cont…
Dried
Mycelium
Direct
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

Molecular Analysis

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

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”

Tree Crown Rating
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
A C
B

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
Epicoccum nigrum
Endophyte

Cont…
Recent Region of occurrence
Wilcox, West Crenshaw County (AL)
Symptoms – Yellow banding, defoliation &
dying trees
Pathogen - Brown-spot fungi?

Cont…
Upson (GA)
Hampton (SC)
(3 infected stands)
Symptoms
Brown needles at the tip, death of the
terminal buds, needle defoliation
Diplodia tip blight, Diplodia sapinea
Diplodia seriata
Trichoderma caerulescens
Hormonema macrosporum
Endophyte

Cont…
Bibb (AL)
Ware & Camden (GA)
(3 infected stands)
Symptoms
Dead and dying needles, football shaped
fruiting bodies, needle defoliation
Lophodermium needle cast, Lophodermium
spp.
Sydowia polyspora
Hendersonia pinicola
Endophyte

Cont…
Macon & Colbert (AL)
Nachitoches (LA)
(4 infected stands)
Symptoms
White aecia, browning needles,
defoliation
Pine needle rust, Coleosporium spp.
Brown-spot needle blight, L. acicola
Epicoccum nigrum
Fungal endophyte
Endophyte

Fungal Pathogens
Fig. 6 Disease symptoms and reproductive structures of (A) L. acicola; (B) Coleosporium sp.;
(C) Lophodermium sp. & (D) D. sapinea on loblolly pine
A
D
C
B

L. acicola Phylogenetic Tree
Fig. 7 Maximum likelihood (ML) tree representing L. acicola and three lineages of L. acicola
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

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

Needle Fungi
Fig. 8 NJ tree representing pathogenic, endophytic & saprophytic fungi found in the needles
Pathogen
52%
Endophyte
24%
Saprophyte
24%
Pathogen
Endophyte
Saprophyte

Frequency of Fungal Pathogens
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
Pathogen
prevalence
Fig. 9 Pathogen prevalence of each species including L. acicola, Lophodermium
sp., Coleosporium sp., D. sapinae and all species combinations
0%
10%
20%
30%
40%
50%
60%
Individual
species
Two species Three
Species
Disease
Incidence
LA & RK
& CS
LP & CS
LA & CS
LA & RK
Diplodia
sapinea
Coleospo
rium sp.
R.
kalkhoffii
Lophoder
mium sp.

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

Loblolly Pine Health Monitoring
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Stand 1 Stand 2 Stand 3 Stand 4 Stand 5 Stand 6 Stand 7
Proportion
of
crown
affected
Chlorosis
Year 2019
Year 2020
P < 0.0001
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)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Stand 1 Stand 2 Stand 3 Stand 4 Stand 5 Stand 6 Stand 7
Proportion
of
crown
affected
Defoliation
Year 2019
Year 2020
P < 0.003

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

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.
Fig. 14 Mean temperature around 27 infected stands in the southeastern US from 1980-2017
15.5
16
16.5
17
17.5
18
18.5
19
1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017
Mean Temperature (oC)

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

Acknowledgements
Lab managers
Tina Ciaramitaro
Angelica Baker
Luis Mendez
Graduate Students
Sylvester Menanyih
John Mensah
Jessica Ahl
Needle Shooters
Dr. Lori Eckhardt
Kris Bradley
D.R. Stallworth
John Gunter
Nathan Baker
Micah Walker
Forrest Fay
Committee members
Dr. Lori Eckhardt
Dr. Scott Enebak
Dr. Jeffrey Coleman
Forest Health
State Cooperative

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