1. PLANT HEALTH PROGRESS  Vol. 16, No. 3, 2015  Page 115
Plant Health Research
Association of Diaporthe longicolla with Black Zone Lines
on Mature Soybean Plants
Taylor R. Olson, Ahmed Gebreil, and Ana Micijevic, Department of Plant Sciences, South Dakota State University, Brookings 57007;
Carl A. Bradley, Department of Crop Sciences, University of Illinois, Urbana 61801 (current address: Department of Plant Pathology,
University of Kentucky Research and Education Center, Princeton 42445); Kiersten A. Wise, Department of Botany and Plant Pathology,
Purdue University, West Lafayette, IN 47907; Daren S. Mueller, Department of Plant Pathology and Microbiology, Iowa State University,
Ames 50011; Martin I. Chilvers, Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and
Febina M. Mathew, Department of Plant Sciences, South Dakota State University, Brookings 57007
Accepted for publication 23 August 2015. Published 28 August 2015.
ABSTRACT
Olson, T. R., Gebreil, A., Micijevic, A., Bradley, C. A., Wise, K. A., Mueller, D. S., Chilvers, M. I., and Mathew, F. M. 2015. Association of Diaporthe
longicolla with black zone lines on mature soybean plants. Plant Health Progress doi:10.1094/PHP-RS-15-0020.
In 2014, during a survey for soybean (Glycine max L.) diseases in
Illinois, Indiana, Iowa, Michigan, and South Dakota, zone lines were
observed on the lower stems of soybean plants. The survey was per-
formed by sampling two to three fields per state. In each field, at least
five plants exhibiting zone lines were collected. Isolations were made
from the zone lines by plating 1-cm pieces on potato dextrose agar. A
total of 90 isolates producing black stromata in concentric patterns and
alpha conidia were tentatively identified as Diaporthe longicolla (Hobbs)
Santos, Vrandecic and Phillips. DNA was extracted from the mycelia of
10 representative isolates and the identity was confirmed by sequencing
the internal transcribed spacer (ITS) region. Additionally, phylogenetic
analysis combining translation elongation factor-1α and actin sequences
was performed and the ten isolates grouped with the D. longicolla ex-type
cultures in a well-supported clade (94% bootstrap support). A patho-
genicity test was performed in the greenhouse by inserting D. longicolla-
infested toothpicks into the lower stems of the soybean plants. To
complete Koch’s postulates, D. longicolla was re-isolated from the zone
lines of the inoculated plants and the pathogen identity was confirmed by
sequencing the ITS gene.
INTRODUCTION
Species of Diaporthe Nitschke cause Phomopsis seed decay,
pod and stem blight, and stem canker of soybean (Glycine max
(L.) Merrill). While Diaporthe longicolla (Hobbs) Santos,
Vrandecic and Phillips (syn. Phomopsis longicolla Hobbs) causes
Phomopsis seed decay, D. sojae Lehman causes pod and stem
blight. Stem canker of soybean is divided into northern stem
canker caused by D. caulivora (Athow and Caldwell) Santos,
Vrandecic and Phillips, and southern stem canker caused by D.
aspalathi Janse van Rensburg, Castlebury and Crous. These
diseases are generally observed on soybean after flowering in the
growing season and can cause yield loss under favorable
conditions (Hartman et al. 1999; Wrather et al. 1997).
Diaporthe spp. primarily have been described based on
morphology and the characteristic disease symptoms they cause
on their hosts. However, given the inter- and intra-species
variability, morphological characteristics are inadequate or
unreliable for differentiation of Diaporthe spp. (Udayanga et al.
2014). Recently, molecular phylogenies, especially those derived
from DNA sequence analyses of the internal transcribed spacer
(ITS) region, translation elongation factor-1α (EF-1α), and actin
(ACT) gene regions, have been used to identify Diaporthe species
(Mathew et al. 2015a; Udayanga et al. 2014; Santos et al. 2011).
In 2014, during a late-season survey of soybean diseases in
Illinois, Indiana, Iowa, Michigan, and South Dakota, zone lines
(thin black lines) that did not form a distinctive shape were
observed on the lower portions of the stems of the mature
soybean plants (Fig. 1). Occasionally, the lines formed irregular
circles of 0.5 to 0.8 mm in diameter. In each state, at least five
FIGURE 1
Zone lines observed inside the lower stem of a soybean plant. (Photo
credit: Febina Mathew and Kiersten Wise).
Corresponding author: F. M. Mathew. Email: febina.mathew@sdstate.edu
doi:10.1094/PHP-RS-15-0020
© 2015 The American Phytopathological Society

2. PLANT HEALTH PROGRESS  Vol. 16, No. 3, 2015  Page 116
plants were collected near or after maturation from two to three
arbitrarily selected soybean fields and examined for stems
containing zone lines (Table 1).
A study by Barr (1978) showed that Diaporthe spp. have
“stromatic tissues composed of prosenchymatous hyphae mixed
with substrate tissues in entostromatic regions, that often form
dark marginal zones above in epidermal tissues and frequently
deep in wood beneath perithecia or groups of perithecia.” More
recently, zone lines were characterized as a symptom produced by
either Macrophomina phaseolina (Tassi) Goid, Fusarium spp., or
Diaporthe spp. on soybean (Ghissi et al. 2014; Zaccaron et al.
2014). The objective of this study was to establish the identity of
the Diaporthe spp. associated with black zone lines produced on
the lower stems of mature soybean plants in Illinois, Indiana,
Iowa, Michigan, and South Dakota.
ISOLATION AND IDENTIFICATION OF THE CAUSAL AGENT
Stem samples (approximately 90) collected from the five states
were washed in tap water for 2 min; and approximately 1 cm-long
pieces were cut from the zone lines of the infected stems. The
pieces from each stem sample were surface-disinfested in sodium
hypochlorite (0.05%) and then ethanol (70%) for 1 min each,
rinsed in sterile distilled water four times, and blotted between
sterile filter paper. Four pieces were placed on potato dextrose
agar (PDA; Becton, Dickinson and Company, Franklin Lakes, NJ)
acidified to pH 4.5 with 85% lactic acid. Plates were incubated at
25
o
C for 7 to 10 days under fluorescent light with a photoperiod
of 12 h daily. Mycelial colonies appeared white, dense, and
floccose, and large black stromata were formed in concentric
patterns or scattered. Alpha conidia exuded from the pycnidial
ostiole in creamy-to-yellowish drops and were ellipsoid and
biguttulate with an average length of 6.6 μm and width of 2.1 μm.
Beta conidia and perithecia did not form on PDA. Ninety isolates
(one isolate per symptomatic plant) from the five states were
tentatively identified D. longicolla based on these morphological
characteristics (Santos et al. 2011). From the 90 symptomatic
stem samples collected from the five states, only D. longicolla
was isolated from the zone lines on the soybean stems. The 90 D.
longicolla isolates included 3 from Illinois, 20 from Indiana, 30
from Iowa, 30 from Michigan, and 7 from South Dakota.
To perform molecular identification of the causal agent, a total
of 10 representative isolates were selected and DNA was
extracted from the lyophilized mycelium scraped from the surface
of 10 day old cultures growing on PDA using the FastDNA Spin
Kit (MP Biomedicals, Solon, OH) (Table 1). The Diaporthe
isolates were identified to species by amplifying and sequencing
of the internal transcribed spacer (ITS) regions using primers
ITS1 and ITS2 (White et al. 1990). Approximately 600 bp of the
ITS region was amplified from the 10 isolates. Forward and
reverse sequences were assembled into contigs using BioEdit
software (v7.2.5) (Hall 1999). Analysis of the edited sequences
was performed using the Basic Local Alignment Search Tool
Nucleotide (BLASTn) searches at the GenBank database
(National Center for Biotechnology Information, http://www.ncbi.
nlm.nih.gov). A BLASTn search of GenBank performed for the
ITS sequences of the 10 isolates showed that the best match was
P. longicolla (syn. D. longicolla) strain STAM-35 (GenBank
Accession No. AY745021) from soybean in the United States
with identities ranging from 98% to 100%. Phylogenetic analyses
of the ITS sequences using maximum parsimony method placed
the 10 isolates in the group containing isolates of D. longicolla
and D. sojae from soybean in the United States (bootstrap value
of 85%; data not presented).
In order to establish a well-resolved phylogeny and clarify the
phylogenetic position, the 10 isolates were characterized by
phylogenetic analyses of two gene fragments, which included
EF1-α and ACT regions. The intron region of the EF1-α gene was
amplified using primers EF1-728F and EF1-986R (Carbone and
Kohn 1999). For the sequencing of the partial actin gene, frag-
ments of ACT gene were amplified using the primers ACT-512F
and ACT-783R (Carbone and Kohn 1999). All DNA samples were
sequenced (GenScript USA Inc., Piscataway, NJ) using the
respective primers. DNA sequences generated in this study have
been deposited under GenBank Accession Nos. KR815475 to
KR815494 and KT021543 to KT021552 (Table 1).
The EF1-α and ACT sequences of the 10 D. longicolla isolates
were aligned using the default parameters of ClustalX (Thompson
et al. 1997) and adjusted manually by visual examination using
the Molecular Evolutionary Genetics Analysis (MEGA) software
v6 (Tamura et al. 2013) prior to being exported as NEXUS files
for subsequent analyses. The outgroup Chrysoporthella hodges-
iana Gryzenhout and Wingfield (Isolate CMW9995) was obtained
from GenBank (GQ290152 for EF1-α sequence and GQ290170
for ACT sequence). The 45 sequences in the combined data set
(including the outgroup C. hodgesiana) comprised 688 bp of
aligned sequence. Phylogenies based on maximum parsimony
were inferred using MEGA, and heuristic searches for the most
parsimonious trees were conducted using the tree bisection-
TABLE 1
Diaporthe longicolla isolates used in this study, the genes sequenced, and the corresponding GenBank Accession Nos.
Isolatesa,b Locationb Species Identityc
GenBank Accession Numbers
ITS EF1-α ACT
IL1 Champaign County, IL D. longicolla KR815475 KR815485 KT021543
IL2 Champaign County, IL D. longicolla KR815476 KR815486 KT021544
IN1 Knox County, IN D. longicolla KR815477 KR815487 KT021545
IN2 Porter County, IN D. longicolla KR815478 KR815488 KT021546
IA1 Montgomery County, IA D. longicolla KR815479 KR815489 KT021547
IA2 Lee County, IA D. longicolla KR815480 KR815490 KT021548
MI1 Ingham County, MI D. longicolla KR815481 KR815491 KT021549
MI2 Ingham County, MI D. longicolla KR815482 KR815492 KT021550
SD1 Brookings County, SD D. longicolla KR815483 KR815493 KT021551
SD2 Miner County, SD D. longicolla KR815484 KR815494 KT021552
a A total of 10 isolates (two representative isolates from each state) were used for identification.
b
IL = Illinois, IN = Indiana, IA = Iowa, MI = Michigan, and SD = South Dakota.
c
Species identity was established based on sequence analysis of the internal transcribed spacer region (ITS), elongation factor subunit 1-α (EF1-α) and
actin (ACT) gene regions.

3. PLANT HEALTH PROGRESS  Vol. 16, No. 3, 2015  Page 117
regrafting (TBR) algorithm (Nei and Kumar 2000) with search
level 1. The initial trees were obtained by random addition (10
replicates). Gaps were treated as missing data, and relative
support for branches was estimated with 1,000 bootstrap replica-
tions (Felsenstein 1985). Prior to the combined analyses, the
concordance of the two gene datasets was evaluated with the
partition-homogeneity test (Farris et al. 1994) implemented with
PAUP* v4.0b10 (Sinauer Associates, Inc., Sunderland, MA;
Swofford 2002) using 1,000 random repartitions (Felsenstein
1985), and with MAXTREES set to 5,000. The null hypothesis of
congruence was rejected if P < 0.001 (Dettman et al. 2003). For
the combined dataset, sequences were concatenated using
Mesquite v2.75 (Maddison and Maddison 2011).
The EF1-α and ACT genes were combined for characterization
of the 10 isolates based on the results of the partition-homo-
geneity test (P = 0.002), indicating that the EF1-α and ACT trees
reflect the same underlying phylogeny. The maximum parsimony
analysis for the combined dataset resulted in five most-parsi-
monious trees and a consensus tree was inferred from the five
trees (length = 262; Fig. 2). The consistency index, retention
index, and rescaled consistency index were 0.75, 0.92, and 0.74,
respectively. The D. longicolla isolates from the five states
formed a monophyletic group with the ex-type cultures CBS 179
and CBS 180 that was supported by a bootstrap value of 94%
(Fig. 2).
The cultural morphology and DNA sequence analysis of the 10
isolates from the five states corresponded to the description of D.
longicolla (syn. P. longicolla) (Santos et al. 2011).
PATHOGENICITY OF DIAPORTHE LONGICOLLA ISOLATES
The D. longicolla isolates were assayed for pathogenicity using
a modified toothpick inoculation method (Ghissi et al. 2014;
Keeling 1982). Seeds of a commercial soybean cultivar of relative
maturity 1.8 (Monsanto Company, St. Louis, MO) were sown into
a potting mix (Sunshine Mix #1, Sun Grow Horticulture Products,
Belleview, WA) in 7.5-liter, circular, plastic pots, one seed per pot
and grown for 4 weeks at 22°C in the greenhouse under a 14-h
photoperiod with a light intensity of 450 μE/m2
/s and watered
daily. The trial was conducted in a completely randomized design
with six replicates (pots) per treatment (D. longicolla isolate), and
the experiment was repeated once.
To obtain inoculum, the isolates were grown on PDA with
sterilized wood toothpicks placed on top of the media. After a
week of incubation, the toothpicks were removed from the PDA
and inserted 2 to 3 mm deep into the lower stems (above the
cotyledon) of soybean plants that were at approximate develop-
mental stages V3 to V4 (Fehr et al. 1971). The toothpicks and
stems were wrapped with Parafilm to avoid rapid dehydration. Six
plants were inoculated per isolate and one toothpick was inserted
per plant. Six control plants were treated by inserting one sterile,
non-infested toothpick into each stem. After inoculation, plants
were kept in the greenhouse for 7 weeks at 22°C with a 14-h
photoperiod. From the beginning of seed development (R5) plant
growth stage until maturation, the soybean plants were not
watered. Instead, the soybean plants were allowed to dry slowly
in the greenhouse at R5 developmental stage so that the fungal
inoculum on the inoculated plants would be dried in situ. At
developmental stage R7 (beginning maturity), soybean plants
were removed from the pots and washed, and examined for zone
lines on the soybean stems (Fig. 3). Upon examination, it was
observed that the occurrence of zone lines on the soybean stems
varied among the ten D. longicolla isolates. For example, zone
lines appeared on the exterior of the lower stems of the soybean
plants 7 weeks after inoculation with isolate IL1 compared to 8
weeks with the other isolates (Fig. 3). Zone lines were not
observed on any of the control plants.
To complete Koch’s postulates, D. longicolla was re-isolated
from the inoculated soybean plants, and the identity was
confirmed via sequencing of the ITS region using primers ITS1
and ITS2 (White et al. 1990). The pathogen was not recovered
from any of the control plants.
FIGURE 2
Consensus of five most-parsimonious trees (length = 262) resulting
from the alignment of 688 characters of the combined EF1-α and ACT
gene region of the Diaporthe longicolla isolates using the maximum-
parsimony analyses. The consistency index, retention index, and
rescaled consistency index were 0.75, 0.92, and 0.74, respectively.
Bootstrap values greater than 50 are shown. D. longicolla isolates
recovered from soybean fields in the five states have state labels (IL =
Illinois, IN = Indiana, IA = Iowa, MI = Michigan, and SD = South Dakota).
The ex-type cultures CBS 179 and CBS 180 are labelled ‘dl’ for D.
longicolla. Chrysoporthella hodgesiana (isolate CMW9995) was used as
the outgroup (National Center for Biotechnology Information Accession
No. GQ290152 for EF1-α sequence and GQ290170 for ACT sequence).
Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013).

4. PLANT HEALTH PROGRESS  Vol. 16, No. 3, 2015  Page 118
IMPORTANCE AND IMPACT
This study demonstrated that the causal fungus producing zone
lines on the lower stems of mature soybean plants in Illinois,
Indiana, Iowa, Michigan, and South Dakota is D. longicolla. The
identification of the causal agent was confirmed by morphology,
DNA sequence analyses of the ITS, EF-1α, and ACT gene
regions, and completion of Koch’s postulates. The phylogenetic
tree generated using the maximum parsimony method by
combining EF-1α and ACT dataset clustered the D. longicolla
isolates from the five states with the D. longicolla ex-type
cultures CBS 179 and CBS 180 forming a monophyletic group
(94% bootstrap support; Fig. 2). Previously, Ghissi et al. (2014)
identified D. phaseolorum var. sojae to be associated with zone
lines on soybean plants based on morphological characteristics.
Zaccaron et al. (2014) associated D. longicolla, M. phaseolina,
and Fusarium spp. with the zone lines observed on tap roots of
the soybean plants based on the recovery of the three fungal
pathogens from the zone lines. However, in this study, only D.
longicolla was isolated from the zone lines on the soybean stems
sampled from the five states. To the best of our knowledge, this is
the first study demonstrating that D. longicolla is associated with
black zone lines on mature soybean plants in Illinois, Indiana,
Iowa, Michigan, and South Dakota.
In this study, the pathogenicity experiment to produce zone
lines by D. longicolla on soybean plants was performed using a
modified protocol of Ghissi et al. (2014) and Keeling (1982). The
protocol was modified from R5 developmental stage to stop
watering the soybean plants and subject them to slow drying in
the greenhouse; given previous studies suggested that zone lines
may be formed in host tissues from the slow drying of the host
plant (Nikandrow 1989; Hubert 1924). However, no watering
during R5 development stage accelerated the natural senescing of
the soybean plants in this experiment. This also caused the
soybean plants to break off as they approached the R7 develop-
ment stage and the experiment had to be terminated. As a result of
this, zone line and stroma were only observed on the exterior of
the lower stem of inoculated plants (Fig. 3).
In the pathogenicity study, differences were observed among D.
longicolla isolates for the amount of time to produce zone lines
and stroma on soybean stems under greenhouse conditions. For
instance, Isolate IL1 produced zone lines and stroma on the
exterior of the lower stems of soybeans in 7 weeks as compared to
the other isolates in 8 weeks indicating that the D. longicolla
isolates may possibly vary in virulence. Although it is likely that
virulence of the D. longicolla isolates or environmental condi-
tions may have influenced pathogen establishment on the host and
the rate at which the pathogen can grow through the host tissue,
this was beyond the scope of our study. However, recent studies
by Zaccaron et al. (2014) have indicated that the production of
zone lines in senesced soybean plants seems to be affected by
cultivar type and location. Further research is warranted to
understand the role of zone lines during the interaction between
D. longicolla and soybean.
D. longicolla primarily causes Phomopsis seed decay of
soybean in areas where high temperatures and high humidity
occur during crop maturation (Hobbs et al. 1985). In Illinois and
other parts of the North Central United States, Phomopsis seed
decay is known to be endemic on soybeans seeds (Sinclair 1993).
Although the infection of soybean seeds with D. longicolla has
resulted in significant economic losses, especially in the mid-
southern region of the United States (Hepperly and Sinclair
1978), there is very little information on stem diseases caused by
D. longicolla. In North Dakota and South Dakota, soybean stem
canker-affected samples were collected during late-season disease
surveys and D. longicolla was isolated from these stems along
with D. caulivora (Mathew et al. 2015b; A. Gebreil and F.
Mathew, unpublished data). Pathogenicity studies by Gebreil et
al. (2015) suggest that D. longicolla may be more aggressive than
D. caulivora on soybean stems. Although D. longicolla has been
identified as a stem pathogen on soybean in North Dakota and
South Dakota (Mathew et al. 2015b; Gebreil et al. 2015), there is
no information on associated yield loss and seed quality.
In summary, the formation of zone lines on the stems of mature
soybean plants is diagnostic of the presence of D. longicolla.
Previously, zone lines were characterized as produced by the
Charcoal rot pathogen (M. phaseolina), Fusarium spp., or
Diaporthe spp. (Ghissi et al. 2014; Zaccaron et al. 2014; T.
Hughes, personal communication), when they were observed on
soybean as the plants approached maturity. Studies by Nikandrow
(1989) and Campbell (1933) suggest that zone lines are the cross-
sectional view of melanized fungal tissue embedded in the host.
On grapevine (Vitis vinifera L.), canes have zone lines when
perithecia (fruiting bodies) produced by the causal fungus (D.
viticola Nitschke) are present (Scheper et al. 2000). On soybean,
it is not clear if the formation of zone lines is related to the
production of perithecia by D. longicolla and this warrants further
research. Although there is limited information available on the
biology of D. longicolla as a stem pathogen and subsequent
disease development, agricultural personnel and crop consultants
in the soybean industry should be aware that the black zone lines
are produced by D. longicolla on soybean plants.
ACKNOWLEDGMENTS
This research was supported by the South Dakota Soybean Research
and Promotion Council (Sioux Falls, SD), the South Dakota Agricultural
Experimental Station (Hatch Project H527-14), and the North Central
Soybean Research Program (Ankeny, IA). We thank Warren Pierson,
Nolan Anderson, Connie Tande, Kay Ruden, Paul Okello, Luke
Hyronimus, Scott Mages, Brian Kontz, and Alec Weber for their
assistance in this study.
FIGURE 3
Zone lines produced by (A) Diaporthe longicolla on the exterior of the
lower stem of a soybean plant under natural conditions and (B) by D.
longicolla isolate IL1 on the lower stem of a soybean plant in the
greenhouse (Photo credit: Connie Tande).

5. PLANT HEALTH PROGRESS  Vol. 16, No. 3, 2015  Page 119
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