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Fungi
Journal of
Article
Assessment of Inoculation Methods of Thielaviopsis paradoxa
(De Seynes) Höhn into Oil Palm Seedlings under
Greenhouse Conditions
Sandra Gaitán-Chaparro 1, Edwin Navia-Rodríguez 1and Hernán Mauricio Romero 1, 2, *
Citation: Gaitán-Chaparro, S.;
Navia-Rodríguez, E.; Romero, H.M.
Assessment of Inoculation Methods
of Thielaviopsis paradoxa (De Seynes)
Höhn into Oil Palm Seedlings under
Greenhouse Conditions. J. Fungi 2021,
7, 910. https://doi.org/10.3390/
jof7110910
Academic Editor: Katrina
Maria Ramonell
Received: 1 September 2021
Accepted: 20 October 2021
Published: 27 October 2021
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Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
1Colombian Oil Palm Research Center—Cenipalma, Oil Palm Biology and Breeding Research Program,
Bogotá11121, Colombia; slgaitanc@unal.edu.co (S.G.-C.); enavia@cenipalma.org (E.N.-R.)
2Department of Biology, Universidad Nacional de Colombia, Bogotá11132, Colombia
*Correspondence: hmromeroa@unal.edu.co
Abstract:
Oil palm (Elaeis guineensis Jacq. and Elaeis Oleifera Cortes) is one of the most important oil
crops in the world. Colombia is the fourth-largest oil palm producer worldwide. However, oil palm
diseases are a significant factor affecting yield. Thielaviopsis paradoxa (De Seynes) Höhn is a pathogen
that affects young palm trees, causing spear rot. Four disease establishment methods were studied to
replicate, in a controlled environment, the symptoms of the disease found in the field. Young palm
trees were inoculated with a suspension of endoconidia using either local infiltration, drip, scissor
cut, or direct contact with agar blocks bearing mycelia and conidia. The effects of the inoculation
methods were studied in dose-method-disease severity experiments conducted in a greenhouse under
controlled conditions. All four methods resulted in T. paradoxa infections and the development of
symptoms of the disease. The disease severity was correlated with the method and dose of inoculation.
In trials to test Koch’s postulates, T. paradoxa was isolated from areas of disease progression in the
inoculated trees, but the teleomorph Ceratocystis paradoxa (Dade) Moreau was not observed. A
photographic record of the infection process at different times post-infection was compiled. Given
that establishing the disease through artificial inoculation is essential for assessing plant pathogenesis,
this study determined that the local infiltration method (
1×106
endoconidia mL
−1
) and a 3–7 day
incubation period were critical for the development of symptoms as severe as those observed in
natural infections in the field.
Keywords:
spear leaf rot; inoculation methods; interspecific oil palm hybrid; plant–pathogen interac-
tions; Thielaviopsis paradoxa
1. Introduction
The genus Elaeis belongs to the monocotyledonous family Arecaceae. It comprises two
taxonomically well-defined species, the African oil palm Elaeis guineensis and the American
oil palm Elaeis oleifera. Oil palm is one of the most important oil crops globally, given that
more than 33% of oils and fats consumed worldwide are derived from it. Moreover, oil
palm is the most productive oil crop, yielding approximately 4 tons oil ha
−1
year
−1
, a
figure nearly ten times higher than the yields of other important oil crops [
1
]. In South
America (Ecuador, Brazil, Peru, Venezuela) and Central America (Costa Rica), the primary
barrier to oil palm crop development is the plant’s susceptibility to pests and pathogens,
the control of which can account for up to 30% of production costs [2,3].
One of the pathogens of oil palm is Thielaviopsis paradoxa, the anamorph of Ceratocystis
paradoxa, which belongs to the family Ceratocystidaceae [4]. It is a pathogen with a broad
host range, including economically important crops, such as sugarcane, date palm, banana,
sorghum, cocoa, sweet potato, coconut, pineapple, corn, and other palm species [
5
–
7
].
T. paradoxa can infect any part of the plant, creating a wide variety of symptoms. Diseases
caused by T. paradoxa have a high destructive potential for oil palm plantations. They
J. Fungi 2021,7, 910. https://doi.org/10.3390/jof7110910 https://www.mdpi.com/journal/jof
J. Fungi 2021,7, 910 2 of 11
have been designated in many different ways in the literature (stem exudation, black
burn, spear leaf rot, and bud rot) [
4
,
5
]. These names describe symptoms that may or
may not be expressed in each instance and reflect a particular symptomatology in specific
palm taxa. In Italy, this fungus has been reported as a causal agent of stem rot in Phoenix
dactylifera [
6
,
7
] and Howeia forsteriana [
8
]. The fungus has also been reported as the causal
agent of bunch discoloration disorder in most date palm-producing countries, including
Saudi Arabia [
9
–
11
], Iraq, Kuwait [
12
], Qatar [
13
], and Egypt [
14
]. Additionally, this fungus
has been reported as the causal agent of bud rot in Hyophorbe lagenicaulis (bottle palm
tree) in Thailand [
15
], as well as in other palm species, such as Areca catechu,Hyophorbe
lagenicaulis, Phoenix africanus,Rhapis sp., Roystonea elata,Sabal palmetto,Syagus romanzoffiana,
and Washingtonia filifera [
4
,
16
,
17
]). Additionally, the pathogen has been described as
the causal agent of basal stem rot in ornamental oil palms in China [
18
]. T. paradoxa is
considered an aggressive and difficult-to-control pathogen that can be dispersed by rain,
wind, wounds, and the tools used to harvest or eradicate diseased plants [
5
,
7
,
19
]. Several
studies have been conducted to control this pathogen with biological and chemical methods
in different hosts [6,17,18]
In Colombia, T. paradoxa has been associated with both spear leaf (youngest leaf) rot
and bud rot. In spear leaf rot, this pathogen results in a significant disease in the neotropics
that affects young trees. The initial stage is characterized by the onset of small necrotic
areas on the spear leaf that extend throughout the leaf until it collapses. In some cases, this
rot reaches the meristem, causing the death of the palm. In other cases, the palm controls
the infection. As a result, the palm begins a recovery process, with the emission of small
and deformed spear leaves at the beginning, until reaching the emission of normal spear
leaves [
19
]. In bud rot, a disease of the oil palm caused by Phytophthora palmivora that
destroys the terminal bud and adjacent leaves resulting in tree death, T. paradoxa behaves
like an opportunistic agent [
3
]. In some regions of America, the planting of African oil
palm cultivars (E. guineensis) is limited by the bud rot complex.
For this reason, oil palm breeders became interested in the American oil palm (E. oleifera)
because it is a source of many economically valuable traits, such as resistance to several
diseases, slow height increment, and improved oil nutritional value [
20
]. Thus, interspecific
O
×
G (E. oleifera
×
E. guineensis) hybrids were developed. However, to date, the interaction
O×G hybrids-T. paradoxa has not been characterized.
The objective of this work was to assess four methods for the inoculation of oil palm
with T. paradoxa under controlled conditions to set up a T. paradoxa-oil palm pathosys-
tem that may be used in omics studies. First, we evaluated de inoculation methods on
E. guineensis and then on O
×
G hybrids. The selected methods have been described in the
literature for Thielaviposis basicola and Alternaria brassicae on detached leaves and adapted
to greenhouse conditions [21,22].
The development of these methods will allow researchers to conduct pathogenesis
studies of T. paradoxa in E. guineensis and the interspecific O
×
G hybrids, which would
lead to the characterization of the infection process and the evaluation of the susceptibility
of oil palm genotypes during their interaction with the fungus.
2. Materials and Methods
2.1. Plant Materials
Two types of cultivars were considered for this experiment: (i) Six-month-old E. guineen-
sis (tenera) nursery plants and (ii) four-month-old O
×
G hybrid nursery plants. The E.
guineensis cultivars were used to evaluate T. paradoxa inoculation methods in a susceptible
material. The second type corresponded to hybrids selected as promissory. These hybrids
showed excellent agronomic and productive performance and good behavior against dis-
eases and pests. All the plants were kept in polyethylene bags, 15 cm in diameter and
18 cm in height, and placed in a greenhouse at the Unipalma plantation in Cumaral, Meta
(geographic coordinates are latitude: 4.271
◦
, longitude:
−
73.487
◦
, and elevation: 412 m)
during the whole experiment. The bags were filled with a mix of soil: vermiculite 3:1.
J. Fungi 2021,7, 910 3 of 11
The region has an average annual rainfall of 2800 mm, with a relative humidity of 80%,
a luminosity of 1482 h, an average temperature of 27
◦
C, a minimum of 20
◦
C, and a
maximum of 32 ◦C.
2.2. Fungal Inoculum
The fungal isolate used in this study belongs to a collection of lyophilized strains
of T. paradoxa collected in different regions of Colombia from E. guineensis palms with
varying degrees of severity of spear leaf rot. During previous studies, the strains were
identified at the molecular level (sequences of the internal transcribed spacer regions (ITS)
were used) [
23
]. Their pathogenicity was evaluated on oil palm leaf fragments using 0.4%
agar with T. paradoxa mycelium and establishing the total percentage of tissue invaded
for 15 days. With the results, the strains were categorized according to aggressiveness
(low < 40%, intermediate 40–60%, and high > 60% of invaded tissue). Aggressiveness was
expressed as the percentage of external and internal tissues invaded by the fungus at the
end of the observation time. Differences among strains were analyzed using the Tukey test,
and the strain producing the highest disease severity was selected for the present study.
Four inoculation methods were assessed using two inoculum types. The inocula were
prepared using monosporic isolates: (i) agar block and (ii) spore suspension (endoconidia).
After obtaining T. paradoxa mycelium on potato dextrose agar (PDA) and growing it at
ambient temperature (22
◦
C to 24
◦
C), blocks of agar (0.5 cm square) were cut from the
periphery of a 7-day culture. The endoconidial suspension was prepared by adding 10 mL
of Tween 80 (0.03% in distilled water) to the sporulated culture (10 days), and endoconidia
were collected using fiberglass filtration; three concentrations of 1
×
10
4
, 1
×
10
5,
and
1×106endoconidia/mL were established using Neubauer chamber count.
2.3. Inoculation Methodology
Four methods were used to inoculate seedlings with T. paradoxa: drip, infiltration,
cutting, and direct contact with cultured agar blocks. The seedlings were maintained for
15 days under control conditions, monitoring temperature and humidity using a data
logger thermohygrometer. Humidity was held at 80–100%, and the temperature was
maintained at 26–28
◦
C, from the moment of the inoculation. (i) Inoculation with Mycelium
blocks: agar blocks containing mycelium were placed at the base of the third leaf, which
had previously been given a 2 mm diameter round surface wound with a pipette tip. The
agar blocks were covered with parafilm to guarantee humid conditions. Sterile agar blocks
were used as controls. (ii) Local Infiltration: a hypodermic syringe was used for infiltrating
0.1 milliliters of endoconidial suspension at the base of the third leaf. The infiltration
buffer was used as the control. (iii) Cutting of the Leaf: the apex of the third leaf was cut
using scissors previously soaked in the endoconidial suspension. For control, scissors were
soaked in a buffer solution. (iv) Drip Inoculation: one milliliter of endoconidial suspension
was drip-applied at the base of the third leaf, which had previously been given a superficial
wound. A buffer drip was used as a control.
In the dose-response trial, the disease was assessed 15 days after fungal inoculation at
10
4
, 10
5
, and 10
6
endoconidia mL
−1
. Each seedling was considered a replicate, with three
replicates per treatment. The complete experiment was repeated three times.
In the time-disease trial, symptom development was assessed at 0, 24, 48, 72, 96, 120,
240, and 360 h post-infection (hpi).
2.4. Disease Severity
The lesioned area on the leaf was measured at each post-inoculation assessment time
point. The severity of the disease in each of the thirty O
×
G palms was recorded. The
disease severity was estimated using the Chiang et al. [24] formula (Equation (1)):
Disease Severity = Mean area of the affected tissue/Mean of the total spear-leaf area ×100 (1)
Severity grades were assigned according to the following scores:
J. Fungi 2021,7, 910 4 of 11
0 = No lesion;
1 = Presence of wet lesions;
2 = Presence of necrotic lesions;
3 = Leaf necrosis of less than 50%;
4 = Leaf necrosis of more than 50%;
5 = Necrosis of a large portion of the leaf, leading to the death of the leaf.
The effect of each treatment on the disease severity or the dose-time relationship was
analyzed by one-way analysis of variance (ANOVA) using the XLSTAT software (version
2015.5, Addinsoft Inc., New York, NY, USA). Differences between methods were analyzed
using Fisher’s least significant difference test.
2.5. Assessment of the Infection Process
O
×
G hybrids were used to evaluate the four inoculation methods and the infection
process at the microscopic scale. Altogether, ten post-inoculation times were assessed (6,
12, 18, 24, 48, 72, 96, 120, 240, and 360 hpi), and three replicates were included each time.
The whole experiment was repeated three times. The leaf tissue was treated with 10%
KOH at 90
◦
C for 15 min to remove the cytoplasmic content while preserving cell walls,
enhancing the ability to view fungal structures under the microscope using ink staining.
The sampling of the leaf tissue was destructive.
Additionally, samples from the lesion progression area (area with diseased and healthy
tissue in proportion 30–70) were taken to prove Koch’s postulates. The small tissue cuts
(1–2 cm long) were disinfected with 10% sodium hypochlorite for 1 min, followed by
washing with sterile distilled water, and transferred to an isolation medium (PDA) and
incubated at room temperature (22 ◦C to 24 ◦C).
3. Results
3.1. Thielaviopsis sp. Isolation
The fungus Thielaviopsis sp. was isolated from the leaf of symptomatic nursery O
×
G
hybrids and was morphologically characterized. T. paradoxa forms thick-wall spores and
chlamydospores that produce infectious asexual spores or conidia. Two types of conidia
were observed in fresh preparations from culture and infected tissue; the first was small,
cylindrical, hyaline to pale brown microconidia, 6.3–10.7
×
2.3–4.1
µ
m in size. The second
type was notably larger (11.8–16.2
×
5.9–8.6
µ
m), brown in color, oval (Chlamydospores).
They were responsible for the black color in the final stage of the disease development.
The conidiophores observed were straight, hyaline-colored, with conidia forming in chains
(Figure 1). The morphological characteristics coincide with the previous descriptions of
Thielaviopsis paradoxa (De Seynes) Höhn [21].
J. Fungi 2021, 7, x FOR PEER REVIEW 5 of 11
Figure 1. Microscopic characteristics of Thielaviopsis sp. isolated from infected tissue. The photographs show septate hy-
aline hyphae, microconidia, chlamydospores, and conidiophores. The red bar corresponds to 20 mm.
3.2. Comparison of Inoculation Methods
Inoculations of E. guineensis seedlings with T. paradoxa were successful. The symp-
toms were developed with all four inoculation methods in plants kept for 15 days under
controlled conditions. Although all E. guineensis seedlings showed progressive symptoms
of the disease, the morphology of the lesions varied in accordance with the inoculation
method used. In plants inoculated using cutting and mycelium block, necrosis began at
the injury site and advanced slowly towards the leaflets. With local infiltration and drip
of the endoconidial suspension, wet necrotic lesions developed around the inoculation
site and advanced through the petiole towards the spear leaf. Disease symptoms caused
by infiltration and drip were similar to those observed in the field and described in other
studies [15,19].
The onset and lesion progression over time depended on inoculum dose and the in-
oculation method. Infiltration resulted in the shortest incubation period (48 h) and the
fastest rate of lesion progression. The effect of inoculum concentration on disease severity
was studied for each inoculation method, with lesion progression only being detected at
concentrations of 10
5
and 10
6
(Figure 2). Variance analysis for the severity and assessment
time interaction showed a significant difference for the infiltration method (p = 0.003). Sta-
tistical differences were significant for the concentration of the inoculum used in the in-
jection and drip methods (p = 0.020), leading to the continuation of the experiments with-
out using the 1 × 10
4
concentration.
Figure 2. Inoculation method comparison. E. guineesis disease severity in seedlings inoculated with
different concentrations of T. paradoxa spores (1 × 10
4
, 1 × 10
5,
and 1 × 10
6
endoconidia per milliliter).
Figure 1.
Microscopic characteristics of Thielaviopsis sp. isolated from infected tissue. The pho-
tographs show septate hyaline hyphae, microconidia, chlamydospores, and conidiophores. The red
bar corresponds to 20 µm.
3.2. Comparison of Inoculation Methods
Inoculations of E. guineensis seedlings with T. paradoxa were successful. The symp-
toms were developed with all four inoculation methods in plants kept for 15 days under
controlled conditions. Although all E. guineensis seedlings showed progressive symptoms
J. Fungi 2021,7, 910 5 of 11
of the disease, the morphology of the lesions varied in accordance with the inoculation
method used. In plants inoculated using cutting and mycelium block, necrosis began at
the injury site and advanced slowly towards the leaflets. With local infiltration and drip
of the endoconidial suspension, wet necrotic lesions developed around the inoculation
site and advanced through the petiole towards the spear leaf. Disease symptoms caused
by infiltration and drip were similar to those observed in the field and described in other
studies [15,19].
The onset and lesion progression over time depended on inoculum dose and the
inoculation method. Infiltration resulted in the shortest incubation period (48 h) and the
fastest rate of lesion progression. The effect of inoculum concentration on disease severity
was studied for each inoculation method, with lesion progression only being detected at
concentrations of 10
5
and 10
6
(Figure 2). Variance analysis for the severity and assessment
time interaction showed a significant difference for the infiltration method (p= 0.003).
Statistical differences were significant for the concentration of the inoculum used in the
injection and drip methods (p= 0.020), leading to the continuation of the experiments
without using the 1 ×104concentration.
J. Fungi 2021, 7, x FOR PEER REVIEW 5 of 11
Figure 1. Microscopic characteristics of Thielaviopsis sp. isolated from infected tissue. The photographs show septate hy-
aline hyphae, microconidia, chlamydospores, and conidiophores. The red bar corresponds to 20 mm.
3.2. Comparison of Inoculation Methods
Inoculations of E. guineensis seedlings with T. paradoxa were successful. The symp-
toms were developed with all four inoculation methods in plants kept for 15 days under
controlled conditions. Although all E. guineensis seedlings showed progressive symptoms
of the disease, the morphology of the lesions varied in accordance with the inoculation
method used. In plants inoculated using cutting and mycelium block, necrosis began at
the injury site and advanced slowly towards the leaflets. With local infiltration and drip
of the endoconidial suspension, wet necrotic lesions developed around the inoculation
site and advanced through the petiole towards the spear leaf. Disease symptoms caused
by infiltration and drip were similar to those observed in the field and described in other
studies [15,19].
The onset and lesion progression over time depended on inoculum dose and the in-
oculation method. Infiltration resulted in the shortest incubation period (48 h) and the
fastest rate of lesion progression. The effect of inoculum concentration on disease severity
was studied for each inoculation method, with lesion progression only being detected at
concentrations of 10
5
and 10
6
(Figure 2). Variance analysis for the severity and assessment
time interaction showed a significant difference for the infiltration method (p = 0.003). Sta-
tistical differences were significant for the concentration of the inoculum used in the in-
jection and drip methods (p = 0.020), leading to the continuation of the experiments with-
out using the 1 × 10
4
concentration.
Figure 2. Inoculation method comparison. E. guineesis disease severity in seedlings inoculated with
different concentrations of T. paradoxa spores (1 × 10
4
, 1 × 10
5,
and 1 × 10
6
endoconidia per milliliter).
Figure 2.
Inoculation method comparison. E. guineesis disease severity in seedlings inoculated with
different concentrations of T. paradoxa spores (1
×
10
4
, 1
×
10
5,
and 1
×
10
6
endoconidia per milliliter).
The methods used were infiltration (blue), cutting with scissors (green), and drip (orange). Values
correspond to the means of nine replications at 240 hpi. Error bars correspond to mean standard error.
Interspecific O
×
G hybrid seedlings were inoculated by the previously evaluated
methods in E. guineensis. Seedlings were observed for initial lesions every 24 h. The
progression of lesions was monitored over two weeks and classified in accordance with the
degree of severity of the disease. The development of a small necrotic area surrounded by
a yellow ring was observed at 72 h in the spear leaf base of seedlings inoculated using the
infiltration approach (1
×
10
6
). At 96 hpi, seedlings inoculated using agar block and drip
methods showed the same type of initial lesion. With the scissor cut method, lesions were
observed only after 96 h (Figure 3).
J. Fungi 2021,7, 910 6 of 11
J. Fungi 2021, 7, x FOR PEER REVIEW 6 of 11
The methods used were infiltration (blue), cutting with scissors (green), and drip (orange). Values
correspond to the means of nine replications at 240 hpi. Error bars correspond to mean standard
error.
Interspecific O × G hybrid seedlings were inoculated by the previously evaluated
methods in E. guineensis. Seedlings were observed for initial lesions every 24 h. The pro-
gression of lesions was monitored over two weeks and classified in accordance with the
degree of severity of the disease. The development of a small necrotic area surrounded by
a yellow ring was observed at 72 h in the spear leaf base of seedlings inoculated using the
infiltration approach (1 × 10
6
). At 96 hpi, seedlings inoculated using agar block and drip
methods showed the same type of initial lesion. With the scissor cut method, lesions were
observed only after 96 h (Figure 3).
Figure 3. Disease severity in O × G hybrid seedlings inoculated with T. paradoxa suspensions (1 × 10
5
and 1 × 10
6
endoconidia mL
−1
) using local infiltration, scissor cutting, drip, and agar blocks. In all,
nine replicates were included for each time. No increased disease severity was observed after 240
hpi with any of the methods.
Necrotic lesions ascending from the base towards the end of the leaf were observed
at 120 hpi for the infiltration method (with the two inoculum concentrations) and the drip
method (with the 1 × 10
6
concentration only). Lesions in the spear leaf were present at 240
hpi. The leaves showed varying degrees of severity, with necrosis and spear leaf collapse
evident only with the injection of inoculum at a concentration of 1 × 10
6
. The final obser-
vation was made 15 days following inoculation, at which time there was no evidence of
further lesion progression in any of the methods under assessment (Figure 2).
Tukey’s multiple comparison test with a 95% confidence level in the interaction
method–inoculum concentration showed that the highest percentages of damage oc-
curred with the infiltration method (1 × 10
6
endoconidia mL
−1
), which was statistically dif-
ferent from the rest of the treatments (B). With this method, more than 50% affection was
observed in the spear leaf. A second group (AB) consisted of the infiltration methods with
1 × 10
5
endoconidia mL
−1
and the dripping at the base of the spear leaves with 1 × 10
6
endoconidia mL
−1
(Table 1).
Figure 3.
Disease severity in O
×
G hybrid seedlings inoculated with T. paradoxa suspensions (
1×105
and 1
×
10
6
endoconidia mL
−1
) using local infiltration, scissor cutting, drip, and agar blocks. In all,
nine replicates were included for each time. No increased disease severity was observed after 240 hpi
with any of the methods.
Necrotic lesions ascending from the base towards the end of the leaf were observed
at 120 hpi for the infiltration method (with the two inoculum concentrations) and the
drip method (with the 1
×
10
6
concentration only). Lesions in the spear leaf were present
at 240 hpi. The leaves showed varying degrees of severity, with necrosis and spear leaf
collapse evident only with the injection of inoculum at a concentration of 1
×
10
6
. The final
observation was made 15 days following inoculation, at which time there was no evidence
of further lesion progression in any of the methods under assessment (Figure 2).
Tukey’s multiple comparison test with a 95% confidence level in the interaction
method–inoculum concentration showed that the highest percentages of damage occurred
with the infiltration method (1
×
10
6
endoconidia mL
−1
), which was statistically differ-
ent from the rest of the treatments (B). With this method, more than 50% affection was
observed in the spear leaf. A second group (AB) consisted of the infiltration methods
with
1×105
endoconidia mL
−1
and the dripping at the base of the spear leaves with
1×106endoconidia mL−1(Table 1).
Table 1. Summary of pairwise comparisons for different inoculation methods.
(Tukey’s Honest Significant Difference Test)
Inoculation Method Least Squares Means Groups
Cutting 1 ×1052.500 A
Agar Block 3.000 A
Cutting 1 ×1063.000 A
Drip 1 ×1053.167 A
Drip 1 ×1064.667 A B
Infiltration 1 ×1056.167 A B
Infiltration 1 ×10611.167 B
The analysis of variance determined that the inoculation method that allowed the
reproduction of the symptoms observed in the field and all the stages described in the
J. Fungi 2021,7, 910 7 of 11
severity scale was infiltration with an inoculum concentration of 1
×
10
6
endoconidia mL
−1
.
Although it can be considered a severe method of inoculation due to the damage caused by
the needle, no symptoms or lesions like those caused by T. paradoxa were evident in any of
the controls.
3.3. Infection Characterization
Variance analyses determined that the inoculation method that resulted in symptoms
as severe as those observed in the field was infiltration using an inoculum concentration of
1
×
10
6
endoconidia mL
−1
. The first external evidence of infection was a yellow ring at the
inoculation site, which was observed at 72 hpi; the lesion area increased considerably at
96 hpi. The ring extended along the spear leaf, with a necrotic tissue appearance and spear
leaf collapse at 240 hpi time. However, the results of destructive sampling showed that
lesions were first apparent at 24 hpi, beginning with rot and advancing rapidly along the
immature tissues of the petiole. By 120 h, more than 50% of the petiole was affected with
rot lesions with a fermented smell, resulting in spear leaf collapse at approximately 240 h.
As shown in Figure 4, the external damage caused by the fungus (upper panel) does not
reflect the internal damage during infection (bottom panel).
J. Fungi 2021, 7, x FOR PEER REVIEW 7 of 11
The analysis of variance determined that the inoculation method that allowed the
reproduction of the symptoms observed in the field and all the stages described in the
severity scale was infiltration with an inoculum concentration of 1 × 106 endoconidia mL−1.
Although it can be considered a severe method of inoculation due to the damage caused
by the needle, no symptoms or lesions like those caused by T. paradoxa were evident in
any of the controls.
Table 1. Summary of pairwise comparisons for different inoculation methods.
(Tukey’s Honest Significant Difference Test)
Inoculation Method Least Squares Means Groups
Cutting 1 × 105 2.500 A
Agar Block 3.000 A
Cutting 1 × 106 3.000 A
Drip 1 × 105 3.167 A
Drip 1 × 106 4.667 A B
Infiltration 1 × 105 6.167 A B
Infiltration 1 × 106 11.167 B
3.3. Infection Characterization
Variance analyses determined that the inoculation method that resulted in symptoms
as severe as those observed in the field was infiltration using an inoculum concentration
of 1 × 106 endoconidia mL−1. The first external evidence of infection was a yellow ring at
the inoculation site, which was observed at 72 hpi; the lesion area increased considerably
at 96 hpi. The ring extended along the spear leaf, with a necrotic tissue appearance and
spear leaf collapse at 240 hpi time. However, the results of destructive sampling showed
that lesions were first apparent at 24 hpi, beginning with rot and advancing rapidly along
the immature tissues of the petiole. By 120 h, more than 50% of the petiole was affected
with rot lesions with a fermented smell, resulting in spear leaf collapse at approximately
240 h. As shown in Figure 4, the external damage caused by the fungus (upper panel) does
not reflect the internal damage during infection (bottom panel).
0 hpi 24 hpi 48 hpi 72 hpi 96 hpi 120 hpi 196 hpi 240 hpi
Figure 4.
Disease progression in oil palm hybrid O
×
G seedlings inoculated using local infiltration
of a T. paradoxa suspension of 1
×
10
6
endoconidia mL
−1
. The externally and internally. The
external damage (upper panel) and internal damage (bottom panel) were recorded at different hours
post-infection (hpi).
Following physical contact between T. paradoxa and palm tissue, the infection was
initiated by germination of endoconidia. Germ tubes were first evident after 12 hpi,
and hyphae formation was observed at approximately 18 h. On occasion, these hyphae
connected with hyphae from other endoconidia and extended towards the stomata. At
72 hpi, branching of hyphae within the tissue was first observed, followed by the presence
of endoconidia throughout the necrotic tissue at 96 hpi (Figure 5).
J. Fungi 2021,7, 910 8 of 11
J. Fungi 2021, 7, x FOR PEER REVIEW 8 of 11
Figure 4. Disease progression in oil palm hybrid O × G seedlings inoculated using local infiltration
of a T. paradoxa suspension of 1 × 106 endoconidia mL−1. The externally and internally. The external
damage (upper panel) and internal damage (bottom panel) were recorded at different hours post-
infection (hpi).
Following physical contact between T. paradoxa and palm tissue, the infection was
initiated by germination of endoconidia. Germ tubes were first evident after 12 hpi, and
hyphae formation was observed at approximately 18 h. On occasion, these hyphae con-
nected with hyphae from other endoconidia and extended towards the stomata. At 72 hpi,
branching of hyphae within the tissue was first observed, followed by the presence of
endoconidia throughout the necrotic tissue at 96 hpi (Figure 5).
Figure 5. Infection process of O × G oil palm hybrid seedlings after T. paradoxa inoculation with a 1
× 106 endoconidia mL−1 suspension using the local infiltration method. (A) Healthy oil palm petiole
tissue (10× magnification). (B) Six hpi (10× magnification). (C) Germ tube formation 12 hpi (100×
magnification). (D) Septum formation and hyphae colonization 18 hpi (100× magnification). (E) Hy-
phae connections 24 hpi. (F,G) Hyphae around the stomata (100× magnification). (H,I) Germinated
endoconidium with hyphae advancing towards the stoma (10× and 100× magnification, respec-
tively). (J) Tissue colonization 36 hpi (40× magnification). (K,L) Sporulation process between 76 hpi
and 96 hpi (40× and 10× magnification, respectively). hpi = hours post-inoculation.
4. Discussion
In this work, four inoculation methods were adapted and evaluated to infect oil palm
spear leaves with T. paradoxa. Lesion progression and symptoms in complete plants under
greenhouse conditions were recorded as a requirement for establishing a T. paradoxa-oil
palm pathosystem. Complete plants were preferred because, although detached parts of
plants are the most suitable plant material to inoculate pathogens at a confined level [22],
they could show a series of responses related to the reaction against infection by patho-
gens and general stress and senescence [25]. Furthermore, key signaling pathways could
be lost because of the lack of connection between roots and stem [26].
The inoculation method with the best results was local infiltration with a 1 × 106 co-
nidia per ml suspension. It was the most appropriate method because the area of injury
was minimal and, more importantly, it allowed reproducing all the symptoms observed
in the field. All four inoculation methods produced infection with T. paradoxa and induced
the disease symptoms. However, there were differences in disease progression and sever-
ity depending on the inoculation method. Furthermore, the results obtained with the in-
filtration and drip methods were consistent with those reported by studies of inoculation
of T. paradoxa in whole plants [14,27].
Figure 5.
Infection process of O
×
G oil palm hybrid seedlings after T. paradoxa inoculation with
a 1
×
10
6
endoconidia mL
−1
suspension using the local infiltration method. (
A
) Healthy oil palm
petiole tissue (10
×
magnification). (
B
) Six hpi (10
×
magnification). (
C
) Germ tube formation 12 hpi
(100
×
magnification). (
D
) Septum formation and hyphae colonization 18 hpi (100
×
magnification).
(
E
) Hyphae connections 24 hpi. (
F
,
G
) Hyphae around the stomata (100
×
magnification). (
H
,
I
) Ger-
minated endoconidium with hyphae advancing towards the stoma (10
×
and 100
×
magnification,
respectively). (
J
) Tissue colonization 36 hpi (40
×
magnification). (
K
,
L
) Sporulation process between
76 hpi and 96 hpi (40×and 10×magnification, respectively). hpi = hours post-inoculation.
4. Discussion
In this work, four inoculation methods were adapted and evaluated to infect oil palm
spear leaves with T. paradoxa. Lesion progression and symptoms in complete plants under
greenhouse conditions were recorded as a requirement for establishing a T. paradoxa-oil
palm pathosystem. Complete plants were preferred because, although detached parts of
plants are the most suitable plant material to inoculate pathogens at a confined level [
22
],
they could show a series of responses related to the reaction against infection by pathogens
and general stress and senescence [
25
]. Furthermore, key signaling pathways could be lost
because of the lack of connection between roots and stem [26].
The inoculation method with the best results was local infiltration with a 1
×
10
6
coni-
dia per ml suspension. It was the most appropriate method because the area of injury was
minimal and, more importantly, it allowed reproducing all the symptoms observed in the
field. All four inoculation methods produced infection with T. paradoxa and induced the
disease symptoms. However, there were differences in disease progression and severity
depending on the inoculation method. Furthermore, the results obtained with the infiltra-
tion and drip methods were consistent with those reported by studies of inoculation of
T. paradoxa in whole plants [14,27].
The results with agar disc and cutting methods were different from other studies.
The agar disc method was modified because it was considered a very aggressive method,
making a punch wound of 5 mm in diameter and 5 mm deep to inoculate the PDA disc
with mycelium and endoconidia, as described previously [
14
,
15
,
27
]. The cutting method
with scissors impregnated with an endoconidia suspension had the drawback of causing
considerable variation in spore distribution, precluding the determination of a mean
inoculum concentration and volume, in addition to producing an injury of significant size.
Although, in most methods, lesions are used in artificial inoculations, they should be mild
because if a response is being evaluated at the molecular level, it will not be clear whether
that response is due to the invasion of the fungus or the intensity of the wound.
J. Fungi 2021,7, 910 9 of 11
Block and cut inoculation methods produced lower severity levels of disease, possibly
because the inoculum was less, and plant defense response is rapidly induced in tissues
with large injured areas [
22
]. Although disease progression with the drip method was
initially rapid, the final severity levels were low, and lesions remained confined to less
than 25% of the tissue. The local infiltration method appears to be the most adequate
considering that the volume of the inoculated pathogen suspension is known and the
injury area is small.
The disease establishment and symptom manifestations were correlated with pathogen
inoculum. The higher the inoculum concentration, the higher the infection. As the inocu-
lum concentration increased, the disease symptoms appeared rapidly. On the contrary, the
disease developed slowly at low inoculum concentrations, and the symptoms appeared late
with no evidence of progression. This correlation between disease severity and inoculum
concentration has been observed in studies of T. basicola [
28
]. The external symptoms were
evident at 72 hpi, with a slight yellow halo at the inoculation site. The symptoms spread
along the spear leaf, with the appearance of necrotic tissue and the collapse of the spear
leaf at 240 hpi. However, inside the seedlings, the affected tissue was observed at 24 hpi.
The characterization of the infection process allowed the establishment of the time
it took to develop the pathogen structures that invaded the host tissue; 12 h for conidia
germination, 24 h for hyphae formation, 48 h for hyphae growth and interconnection,
72 h for tissue invasion, and 96 h for endoconidia formation. These results are of great
interest in omics studies to determine the sampling times according to the focus of the
research. The observation of interconnected hyphae near to stomata suggests that, like other
phytopathogens, T. paradoxa penetrates through natural openings such as stomata [
14
,
29
].
Different ways of entry include mechanical wounds or those caused by insects that feed on
the palm, such as Rhynchophorus palmarum, which has been postulated as a probable vector
of the fungus. This hypothesis is reinforced because T. paradoxa is present in the insect’s
digestive tract [30].
The seedling tissue cuts showed that an exogenous signal was required for T. paradoxa
germ tube development to initiate, which occurred at approximately 12 hpi. This period
was more extended than that observed for T. basícola in tobacco root, which was reported
tobe8h[26].
5. Conclusions
Establishing a disease using artificial inoculations is essential for pathogenesis studies
in plants. In our research, local infiltration with an inoculum concentration of
1×106
en-
doconidia mL
−1
and an incubation period of 3 to 7 days were determinant factors for
seedlings to develop symptoms as severe as those observed in the field. Establishing this
method allowed the observation of the development stages of the disease, which supported
the definition of a severity scale. Establishing the disease by artificial inoculation was
essential to determine an infection process timeline, the first step for sampling in RNASeq
studies during plant-pathogen interaction. Furthermore, the infection process timeline
could be used to study etiology, disease resistance, and disease control. Finally, the infiltra-
tion method could be used in other species of the Arecaceae family affected by T. paradoxa,
adjusting the inoculum concentration.
Author Contributions:
Conceptualization, H.M.R.; Methodology, H.M.R., S.G.-C., E.N.-R.; Formal
analysis, H.M.R., S.G.-C., E.N.-R.; Investigation, H.M.R., S.G.-C., E.N.-R.; Writing—original draft,
H.M.R., S.G.-C.; Writing—review and editing, H.M.R., S.G.-C. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was funded by the Colombian Oil Palm Promotion Fund (FFP) administered
by Fedepalma.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
J. Fungi 2021,7, 910 10 of 11
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy restrictions.
Acknowledgments:
The authors would like to thank the Biology and Breeding Program of Ceni-
palma. The authors would also like to thank Unipalma S.A for the plants and facilities.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
References
1.
Fedepalma. Statistical Yearbook 2019; Federación Colombiana de Cultivadores de Palma de Aceite: Bogotá, Colombia, 2020; p. 237.
2. De Franqueville, H. Oil palm bud rot in Latin America. Exp. Agric. 2003,39, 225–240. [CrossRef]
3.
Torres, G.; Sarria, G.; Martinez, G.; Varon, F.; Drenth, A.; Guest, D. Bud Rot Caused by Phytophthora palmivora: A Destructive
Emerging Disease of Oil Palm. Phytopathology 2016,106, 320–329. [CrossRef] [PubMed]
4. Garofalo, J.F.; McMillan, R.T. Thielaviopsis diseases of palms. Proc. Fla. State Hortic. Soc. 2004,117, 324–325.
5.
Karampour, F.; Pejman, H. Study on possible influence of pathogenic fungi on date bunch fading disorder in Iran. In Proceedings
of the III International Date Palm Conference, Abu Dhabi, United Arab Emirates, 19–21 February 2006; pp. 431–439.
6.
Abdullah, S.; Asensio, L.; Monfort, E.; Gomez-Vidal, S.; Salinas, J.; Lorca, L.; Jansson, H. Incidence of the two date palm pathogens,
Thielaviopsis paradoxa and T. punctulata in soil from date palm plantations in Elx, South-East Spain. J. Plant Prot. Res.
2009
,49,
276–279. [CrossRef]
7.
Polizzi, G.; Castello, I.; Vitale, A.; Catara, V.; Tamburino, V. First report of Thielaviopsis trunk rot of date palm in Italy. Plant Dis.
2006,90, 972. [CrossRef]
8.
Polizzi, G.; Castello, I.; Aiello, D.; Vitale, A. First report of stem bleeding and trunk rot of Kentia palm caused by Thielaviopsis
paradoxa in Italy. Plant Dis. 2007,91, 1057. [CrossRef]
9.
Al-Onazi, M.; Al-Dahain, S.; El-Ansary, A.; Marraiki, N. Isolation and Characterization of Thielaviopsis paradoxa L-alanine
Dehydrogenase. Asian J. Appl. Sci. 2011,4, 702–711. [CrossRef]
10.
Al-Rokibah, A.; Abdalla, M.; El-Fakharani, Y. Effect of water salinity on Thielaviopsis paradoxa and growth of date palm seedlings.
J. King Saud Univ. Agric. Sci. 1998,10, 55–63.
11.
Molan, Y.; Al-Obeed, R.; Harhash, M.; El-Husseini, S. Decline of date palm offshoots infected with Chalara paradoxa in Riyadh
region. J. King Saud Univ.—Agric. Sci. 2004,16, 79–86.
12.
Suleman, P.C.; Al-Musallam, A.; Menezes, C.A. Incidence and severity of black scorch on date palms in Kuwait. Kuwait J. Sci. Eng.
2001,28, 161–169.
13. Abbas, E.; Abdulla, A. First report of neck bending disease on date palm in Qatar. Plant Pathol. 2003,52, 790. [CrossRef]
14.
Farrag, E.S.; Abo-Elyousr, K.A. Occurrence of some fungal diseases on date palm trees in upper Egypt and its control. Plant
Pathol. J. 2011,10, 154–160. [CrossRef]
15.
Soytong, K.; Pongak, W.; Kasiolarn, H. Biological control of Thielaviopsis bud rot of Hyophorbe lagenicaulis in the field. J. Agric.
Technol. 2005,1, 235–245.
16.
Elliott, M.L. Thielaviopsis Trunk Rot of Palm; Fact Sheet. PP-219; Institute of Food and Agricultural Science, University of Florida:
Gainesville, FL, USA, 2006.
17.
Nelson, S. Stem Bleeding of Coconut Palm; College of Tropical Agriculture and Human Resources, Cooperative Extension Service,
University of Hawaii at Manoa: Honolulu, HI, USA, 2005.
18.
Li, F.-H.; Sun, X.-D.; Niu, X.-Q.; Cao, H.-X.; Yu, F.-Y. First Report of Basal Stem Rot on Oil Palm Caused by Thielaviopsis paradoxa in
Hainan, China. Plant Dis. 2018,102, 2029. [CrossRef]
19.
Paterson, R.; Sariah, M.; Lima, N. How will climate change affect oil palm fungal diseases? Crop Protect.
2013
,46,
113–120
. [CrossRef]
20.
Mozzon, M.; Foligni, R.; Mannozzi, C. Current Knowledge on Interspecific Hybrid Palm Oils as Food and Food Ingredient. Foods
2020,9, 631. [CrossRef]
21.
Hewajulige, I.G.N.; Wijesundera, R.L.C. Thielaviopsis paradoxa, Thielaviopsis basicola (Black Rot, Black Root Rot). In Postharvest
Decay; Elsevier: Amsterdam, The Netherlands, 2014; pp. 287–308.
22.
Giri, P.; Taj, G.; Kumar, A. Comparison of artificial inoculation methods for studying pathogenesis of Alternaria brassicae (Berk.)
Sacc on Brassica juncea (L.) Czern. (Indian mustard). Afr. J. Biotechnol. 2013,12, 2422–2426.
23.
Rodríguez, J.; Vélez, D.; Sarria, G.A.; Torres, G.A.; Noreña, C.; Navia, M.; Romero, H.M.; Varón, F.; Martínez, G. Morphological,
molecular and pathogenic identification of microorganisms associated with bud rot disease of oil palm in Colombia. Fitopatol.
Colomb. 2009,33, 49–56.
24.
Chiang, K.-S.; Bock, C.H.; Lee, I.-H.; El Jarroudi, M.; Delfosse, P. Plant disease severity assessment—How rater bias, assessment
method, and experimental design affect hypothesis testing and resource use efficiency. Phytopathology
2016
,106, 1451–1464. [CrossRef]
25.
Liu, G.; Kennedy, R.; Greenshields, D.L.; Peng, G.; Forseille, L.; Selvaraj, G.; Wei, Y. Detached and attached Arabidopsis leaf
assays reveal distinctive defense responses against hemibiotrophic Colletotrichum spp. Mol. Plant-Microbe Interact.
2007
,20,
1308–1319. [CrossRef]
J. Fungi 2021,7, 910 11 of 11
26.
Hood, M.; Shew, H. Initial cellular interactions between Thielaviopsis basicola and tobacco root hairs. Phytopathology
1997
,87,
228–235. [CrossRef] [PubMed]
27.
Warwick, D.; Passos, E.E. Outbreak of stem bleeding in coconuts caused by Thielaviopsis paradoxa in Sergipe, Brazil. Trop. Plant
Pathol. 2009,34, 175–177. [CrossRef]
28.
Holtz, B.; Weinhold, A. Thielaviopsis basicola in San Joaquin Valley soils and the relationship between inoculum density and
disease severity of cotton seedlings. Plant Dis. 1994,78, 986–990. [CrossRef]
29.
Punja, Z.K. Virulence of Chalara elegans on bean leaves, and host-tissue responses to infection. Can. J. Plant Pathol.
2004
,26,
52–62. [CrossRef]
30.
Da Costa, R.R.; Warwick, D.R.N.; de Souza, P.E.; de Carvalho Filho, J.L.S. Transmissibilidade de Thielaviopsis paradoxa, agente
causal da resinose do coqueiro por Rhynchophorus palmarum.Sci. Plena
2011
,7. Available online: https://www.scientiaplena.org.
br/sp/article/view/139 (accessed on 19 October 2021).
