120

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Anita-Sari et al.

Pelita Perkebunan 38(2) 2022, 120

—

127

Flush Characteristics of Several Cocoa Genotypes Different

in Resistance to Vascular Streak Dieback

Indah Anita-Sari

, Bayu Setyawan

, Agung Wahyu Susilo

Nurhadini Fitri Isnaini

Samsul Paputpungan

, Febrilia Nur’aini

, and Nur Solecha Ruseani

)

Indonesian Coffee and Cocoa Research Institute, Jl. PB. Sudirman No. 90, Jember, Indonesia

Bandung Institute of Technology, School of Life Sciences and Technology, Jl. Ganesa No. 10, Bandung, Indonesia

Gorontalo State University, Jl. Jend. Sudirman No. 6, Kota Tengah Gorontalo, Indonesia

Ghent University, Sint-Pietersnieuwstraat 25, 9000 Ghent, Belgium

Received: 11 January 2022 / Accepted: 19 May 2022

Abstract

Vascular streak dieback (VSD) is one of the main diseases on cocoa that can

cause a decrease in production and even death on susceptible plants. The use of

selection criteria is very important in the selection process at the seedling phase,

young plants and even mature plants in order to support the breeding process

of resistant varieties. The aim of this study was to determine the characteristics

of flush including flush color, stomata characters and duration of flush color

change to green or towards mature leaves as one of the selection indicators for

VSD resistance in cocoa. The research was conducted at Kaliwining Experimental

Station, Indonesian Coffee and Cocoa Research Institute, Jember, Indonesia. Study

of flush color and stomata characters was conducted using a randomized complete

block design consisting of four cocoa genotypes with different levels of resistance,

classified as resistant to VSD disease (Sulawesi 1, PNT 16) and as susceptible

(BCL and BL 50). Each clone was repeated three times and each replication consisted

of three plant samples. Duration of flush color change study was carried out on

six cocoa genotypes as treatments, three genotypes with resistant to VSD (PNT 16,

Scavina 12, Sulawesi 1) and three genotypes as susceptible to VSD (BL 50, BCL,

Criollo 22). Each treatment was repeated three times and each replication consisted

of three plant samples. The results showed that the resistant genotypes showed a

tendency to have lower chlorophyll and anthocyanin content than the susceptible

ones. The stomata character in the resistant genotypes was not different than the

susceptible ones, however, the resistant genotype showed that the stomata density

at flush tended to be lower. The duration of flush color change to mature leaves

(green) in resistant genotypes was significantly faster than susceptible genotypes.

Keywords: duration of flush color change, flush color, stomata characters, Theobroma

cacao L., vascular streak dieback

ISSN: 0215-0212 / e-ISSN: 2406-9574

DOI: 10.22302/iccri.jur.pelitaperkebunan.v38i2.518

121

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Flush characteristics of several cocoa genotypes different in resistant to vascular streak dieback

INTRODUCTION

In Indonesia, vascular streak dieback

(VSD) disease is one of the factors that

causes decreasing of cocoa production. This

disease attacks the xylem of flush tissues

and will show symptoms after three months

of infection. VSD disease not only causes

a decrease in plant productivity, but also

causes death of susceptible plants. Utiliza-

tion of resistant genotypes is the main strategy

in controlling VSD disease effectively and

efficiently (Susilo & Anita-Sari, 2011). The

breeding of planting material which resis-

tant to VSD is carried out through explo-

ration, selection and crossing activities. An

effort to support these plant breeding activi-

ties, it is necessary to have selection criteria

for early detection of the nature of plant

resistance to VSD disease. The use of selec-

tion criteria will be very useful in the selec-

tion process for either the seedling phase,

young plants or even mature plants.

The mechanism of VSD infection through

flush takes time since the fungus begins to

infect until the onset of disease symptoms.

The period of flush color changes to green

or mature leaves maybe related to the nature

of plant resistance in response to this disease.

Leaf flushing in cocoa is controlled both

endogenously and environmentally. If envi-

ronmental stresses are not apparent, it is mainly

under endogenous control (Lahive et al.,

2019). The results of previous studies showed

that leaf stomata character could be used

as a selection indicator for VSD resistance

(Anita-Sari & Susilo, 2014). Stomata is one

of the selection criteria that can be used to

detect the resistance of cocoa plants to VSD

disease (Susilo et al., 2016).

The parasitism relationship between the

host and the pathogen is expressed in terms of

resistance and susceptibility. Plant resistance

reactions can occur before infection (pre

infection) and after infection (post infection)

in form of chemical content or tissue structure

owned by plants to inhibit pathogen repro-

duction (Collingborn et al., 2000; Hulupi,

2008). The internal anatomy and surface

features of the leaves often determine plant

resistance to biotrophic pathogen infection

(Pudjiwati et al., 2013), such as stomata and

trichome may influence disease resistance

(Niks & Rubiales, 2002).

Indications of differences in color character

and duration of color change from flush to

mature marked by a change in color to green

maybe related to resistance to VSD disease

considering that VSD only enters plants

through flush tissue. This study was conducted

to examine the characteristics of flush in-

cluding stomata character, flush color and

duration of flush color change to green or

towards mature leaves on some cocoa geno-

types with different level in VSD resistance.

MATERIALS AND METHODS

Flush Color and Stomata Characteristics

The research was conducted at Kaliwining

Experimental Station, Indonesian Coffee and

Cocoa Research Institute, Jember, Indonesia.

The study used a randomized complete block

design (RCBD) consisting of four cocoa geno-

types with different levels of resistance, classi-

fied as resistant to VSD disease (Sulawesi 1,

PNT 16) and susceptible to VSD (BCL and

BL 50). Each clone was replicated three times

and each replication consisted of three plant

samples. Observation parameters include

flush color and stomatal characters. Color

analysis was carried out by measuring the

content of chlorophyll and anthocyanins

in the flush. The analysis of chlorophyll

and anthocyanins was carried out at Jember

Polytechnic using a destructive method

referring to Sims & Gamon (2002) and Porra

(2002). Stomata analysis was carried out using

122

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Anita-Sari et al.

the replica method referring to Anita-Sari

& Susilo (2013) using transparent nail polish

and stomata removal was carried out on the

lower leaves. The stomata samples were then

observed using a microscope and observed

for number of stomata, width of stomata

opening and density of the stomata. The leaf

samples used were flushes which still red

color with the size 10-15 cm.

Flush Color Change Duration

This study was also conducted at Kaliwining

Experimental Station, Indonesian Coffee and

Cocoa Research Institute. The study used a

completely randomized block design consisting

of six cocoa genotypes as treatment, three

genotypes as resistant genotypes (PNT 16,

Scavina 12, Sulawesi 1) and three suscep-

tible genotypes (BL 50, BCL, Criollo 22). Each

treatment was repeated three times and each

replication consisted of three sample plants.

The duration of flush changes was observed

by marking the newly emerged flush with

a size of 7-10 cm and exposed to direct sun-

light. Observations were made by calculating

the time required for the flush to turn green.

Data Analysis

Data were analyzed using one-way

ANOVA via STAR 2.0.1 (IRRI) and Duncan’s

test was further carried out when a significant

difference was found. The histogram was

illustrated using Graphpad program. Principal

component analysis (PCA) was performed with

a dendogram through R program.

RESULTS AND DISCUSSION

Characteristics of Flush Color and Stomata

The result showed that there were dif-

ferences of anthocyanin and chlorophyll content

in flush between resistant and susceptible cocoa

clones (Figure 1). Sulawesi 1 and PNT 16

which were resistant to VSD showed lower

chlorophyll and anthocyanin content than

susceptible clones (BCL and BL 50). This

result is different from the opinion of Tellez

et al. (2016) that anthocyanins content in

tropical crops protect young leaves from

damage caused by fungal attacks during leaf

development into mature leaves. Plants will

protect themselves from fungal attacks through

the production of phenolic compounds such

as flavonoids that function as anti-fungal

(Queenborough et al., 2013; Cheng et al.,

2018). However, a comprehensive review

on the adaptive function of anthocyanins

concludes that the evidence for a defensive

function against herbivores and pathogens

is not very strong (Schaefer et al., 2008).

The tendency for the high content of

anthocyanins and chlorophyll in the flush of

susceptible clones is related to the inhibition

of chlorophyll in the photosynthesis process

so that it interferes with plant biosynthesis.

Chlorophyll plays a significant role in plant

development (Li et al., 2018), the accumu-

lation of anthocyanins and chlorophyll is

related to the regulation of plant development

(Dai et al., 2016). The purple color component,

anthocyanins, and their combination with

chlorophyll can cause an adjustment of the

ratio of the photosynthetic system and improve

electron transport performance. Anthocyanins

can cover chlorophyll more deeply and inhibit

the photosynthesis process (Cooney et al.,

2015; Li & Martin, 2015).

Leaf color is generally used as a morpho-

logical marker in plant breeding programs

because this character is an inherited character

and can be used in important approaches to

plant breeding programs (Cheng et al., 2018).

The results showed that the anthocyanin content

had a high heritability value and the inheritance

was controlled by two major genes in additive-

dominant-epistatic interaction and polygenes.

The major genes controlling leaf and stem

123

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Flush characteristics of several cocoa genotypes different in resistant to vascular streak dieback

anthocyanin content exhibited high herita-

bility in F2 generation (Jin et al., 2018).

Variations in leaf color patterns are also

important and necessary in the assessment

of plant resistance (Plastino et al., 2006;

Palmer & Mascia, 1980; Zhao et al., 2016).

The results of PCA analysis showed that

there was a clear grouping between resistant

and susceptible clones based on chlorophyll

and anthocyanin content where BL 50 indi-

cated one group with BCL and PNT 16 indi-

cated one group with Sulawesi 1 (Figure 2).

These results indicate that there are different

patterns of anthocyanin and chlorophyll

content in susceptible and resistant cocoa

clones to VSD. Adaptation of plants to the

environment can be induced by the propor-

tion of colors that affect plant development,

limiting the size of biological functions, espe-

cially the photosynthetic system (Kerchev

et al., 2011) and the antioxidant system (Zhang

et al., 2013), so that they are associated and

related to plant resistance, whereas suscep-

tibility is related to plant defense.

The characteristics of stomata in the flush

did not show any difference between resistant

and susceptible clones including its length,

diameter, opening width and stomatal density

(Table 1). The results of the analysis showed

that there was no significant difference in

stomatal flush density between resistant and

susceptible genotypes, but resistant geno-

types tended to have lower stomatal density

than susceptible genotypes. The research

results by Susilo et al. (2016) indicated that

there was a correlation of stomata charac-

teristics on young leaves and VSD disease

resistance. The number of stomata and the

width of stomata openings on old leaves

showed a significant correlation to VSD

disease resistance in cocoa. It showed that

there were differences of stomata charac-

teristics on different leaves age.

The low level of stomata density at flush

provides a smaller opportunity for spores to

enter the plant tissue. The higher the stomata

density, the plants tend to be more susceptible

to disease (Bozoglu & Karayel, 2006). A large

number of stomata will increase the transpira-

Figure 1. Anthocyanin and chlorophyll content in flush of some cocoa genotypes with different

resistance levels

Percentage

Clones

Sulawesi 1

PNT 16

BCL

BL 50

Sulawesi 1

PNT 16

BCL

BL 50

Anthocyanin

Chlorophyll

124

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Anita-Sari et al.

tion process and also affect the capture of CO

from the air, so that the chance of entering

spores together with the air will be higher.

The incubation period is the time from inocu-

lation to the appearance of symptoms. Stomata

density has been used as a selection indicator

in breeding disease resistance in cocoa (Anita-

Sari, 2004; Susilo et al., 2016).

Flush Color Change Duration

The results of the analysis showed that

there was a difference in the duration of the

flush color change to green in the cocoa geno-

types tested. Criollo 22, BCL, and BL 50 clones

required a longer duration and were signifi-

cantly different than Sulawesi 1, Sca 12, and

PNT 16 (Table 2).

The resistant clone group showed a rela-

tively faster duration of flush change compared

to the susceptible clones (Table 2). The

duration of time used to change the flush

color to green in resistant clones ranged from

7-8 days, while for susceptible clones it was

more than 10 days. The short period of time

for flush discoloration indicates the least

opportunity for spores to enter plant tissues.

The length of time required for flush color

change in susceptible plants provides a greater

opportunity for spores to infect the flush so

that the fungus can enter plant tissues.

Disease resistance is related to the behavior

of the disease in infecting plants. In Brassica

plants, high resistance to downy mildew occurs

when the leaves are young compared to old

Figure 2. PCA biplot on anthocyanin and chlorophyll content of four cocoa genotypes

-1

-2

Prin 2

-4 -3 -2 -1 0 1 2

Prin 1

BL-50 BCL

PNT-16

Sulawesi-1

Table 1. Characteristics of stomata of flush in several cocoa clones

Clone Width of stomata (m) Diameter of stomata (m) Stomata apperture width (m) Stomata density m

-2

Sulawesi 1 14.5 a 5.23 a 1.15 a 242 a

PNT 16 14.5 a 4.96 a 1.06 a 317 a

BCL 14.4 a 5.33 a 1.09 a 430 a

BL50 14.3 a 4.99 a 1.06 a 386 a

125

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Flush characteristics of several cocoa genotypes different in resistant to vascular streak dieback

leaves. Susceptibility will stop when the leaves

are old because these conditions no longer

support the growth of the pathogen (Coelho

& Monteiro, 2003). Likewise, other plants

including broccoli, show high susceptibility

to older leaves because leaves that are already

chlorotic are difficult for pathogens to grow

and pathogens can no longer sporulate (Agnola

et al., 2003).

In contrast, Kus et al. (2002) found that

older plants or leaves showed increased

resistance and decreased susceptibility to

pathogens, this form of resistance is often

referred to as age-related resistance (ARR).

This result is similar to that of the resistance

of cocoa plants to VSD, the faster the leaves

turn green (mature) the plants are more

resistant than those with a longer duration

of change. The actual mechanism of ARR

occurs in only a few cases, where the ARR

response is thought to be controlled by a single

resistance gene expressed in mature plants

(Roelfs, 1984). There is a positive correlation

between increased leaf/plant age and glycoline

production (Lazarovits et al., 1980).

The results of cluster analysis based on

flush color duration and VSD scores showed

that there were two groups, namely group I

with longer duration category and high VSD

level, namely Criollo 22, BL 50 and BCL and

group II with fast duration category with low

VSD level, namely Sulawesi 1, Sca 12, and

PNT 16.

CONCLUSIONS

The content of anthocyanins, chlorophyll

and duration of flush color change to mature

can be used as indicators of selection for

Table 2. Analysis of the variance of flush color change duration on several cocoa genotypes

Genotype Duration of flush color change (days) VSD resistance

Criollo 22 14.00 a Susceptible

BCL 10.62 b Susceptible

BL 50 10.11 b Susceptible

Sulawesi 1 8.33 cd Resistant

PNT 16 7.12 d Resistant

Sca 12 7.00 d Resistant

Figure 1. Cluster analysis based on duration of flush color change in some cocoa genotypes

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

Average distance between cluster

PNT 16

Sul 1

Sca 12

BCL

BL 50

Criollo

126

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Anita-Sari et al.

resistance to vascular streak dieback disease

in cocoa. Resistant genotypes tend to have

lower chlorophyll content, flush anthocya-

nins and stomatal density with the duration

of flush color change to mature leaves faster

than susceptible genotypes.

REFERENCES

Agnola, B.; S. Boury; C. Monot; A. Quilevere;

Y. Herve & D. Silue (2003). Evidence

that a leaf-disk test allows assessment

of isolate-specific resistance in Brassica

oleracea crops against downy mildew

(Peronospora parasitica). European

Journal of Plant Pathology, 109,

471–478.

Anita-Sari, I. & A.W. Susilo (2013). Pengembangan

kriteria seleksi karakter berat biji pada

tanaman kakao (Theobroma cacao L.)

melalui pendekatan analisis sidik lintas.

Pelita Perkebunan, 29(3), 174–181.

Anita-Sari, I. & A.W. Susilo (2014). Effect of

genetic and altitude differences on

stomata characters as resistance indi-

cators to vascular streak dieback (VSD)

in cocoa (Theobroma cacao L.). Journal

of Agriculture Science and Technology,

4, 157–163.

Bozoglu, H. & R. Karayel (2006). Investigation

of stomata densities in pea (Pisum

sativum L.) lines/cultivars. Biological

Science, 6(2), 56–61.

Cheng, G.X.; R.J. Li; M. Wang; L.J. Huang; A. Khan;

M. Ali & Z.H. Gong (2018). Variation in

leaf color and combine effect of pigments

on physiology and resistance to whitefly

of pepper (Capsicum annuumL.).

Scientia Horticulturae, 229, 215–225.

Coelho, P.S. & A.A. Monteiro (2003). Expression

of resistance to downy mildew at coty-

ledon and adult plant stages in Brassica

oleracea L. Euphytica, 133, 279–284.

Collingborn, F.M.B; S.R. Gowen & I. Mueller-

Harvey (2000). Investigations in to the

biochemical basis for nematode-resis-

tance in roots of three Musa cultivars

in response to Radopholus similis infec-

tion. Journal Agricultural Food Chemis-

try, 48, 5297–5301.

Cooney, L.J.; H.M. Shaefer; B.A. Logan, B. Cox

& K.S. Gould (2015). Functional signifi-

cance of anthocyanins in peduncles in

Sambucus nigra. Environment Experi-

mental Botany, 119, 18–26.

Dai, L.; D. Wang Xie; X. Zhang; Y. XU; Y. Wang

& J. Zhang (2016). The novel gene

vpPR4-1 from vitis pseudoreticulata

increases powdery mildew resistance

in transgenic Vitis vinifera L. front.

Plant Science, 7, 695.

Hulupi, R. (2008). Identifikasi ras fisiologi

nematoda Radopholus similis Cobb.

yang menyerang tanaman kopi. Pelita

Perkebunan, 22(3), 213–221.

Jin, L.; M.J. Qin & Q. Wei (2018). Inheritance

of anthocyanin contents in leaf and

stem of Brassica campestriss ssp.

chinensis var. purpuraria. Journal of

Norhwest A & F University-Natural

Science, 46(3), 119–127.

Kerchev, P.I.; B. Fenton; C.H. Foyer & R.D. Hancock

(2011). Plant responses to insect herbivory:

Interactions between photosynthesis,

reaction exygen species and hormonal

signaling pathway. Plant, Cell, & Envi-

ronment, 35(2), 441–453.

Kus, J.V.; K. Zaton; R. Sarkar & R.K. Cameron

(2002). Age-related resistance in Arabidopsis

is a developmentally regulated defense

response to Pseudomonas syringae.

Plant Cell, 14, 479–490.

Lahive, F.; P. Hadley & A.J. Diamond (2019).

The physiologycal responses of cacao

to the environment and the implications

for climate change resilance. A Review.

Agronomy for Sustainable Development,

39(1), 5.

Lazarovits, G.; C.H. Unwin & E.W.B. Ward (1980).

A rapid assay for systemic fungicides

against Phytophthora rot soybeans. Plant

Disease, 64, 163–165.

Li, Y.C.C. & E. Martin (2015). Leaf anthocyanin,

photosynthetic light use efficiency,

127

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Flush characteristics of several cocoa genotypes different in resistant to vascular streak dieback

and ecophysiology of the South African

succulent Anacampseros rufescens

(Anacampserotaceae). South African

Journal of Botany, 99, 122–128.

Li, Y.; N. He; J. Huo; L. Xu; C. Liu; J. Zhang;

Q.F. Wang; X. Zhang & X. Wu (2018).

Factor influencing leaf chlorophyll

content in natural forest at the biome

scale. Frontiers in Ecology and Evolu-

tion, 6(64), 1–10.

Niks, R.E. & Rubiales (2002). Potentially durable

resistance mechanisms in plants to

specialized fungal pathogens. Euphytica,

124, 201–216.

Plastino, E.M.; S. Ursi & M.T. Fujii (2006). Color

inheritance, pigment characterization,

and growth of a rare light green strain

of Gracilaria birdiae (Gracilariales,

Rhodophyta). Physiological Research,

52(1), 45–52.

Porra, R.J. (2002). The chequered history of the

development and use of simultaneous

equations for the accurate determina-

tion of chlorophylls a and b. Photo-

synthesis Research, 73, 149–156.

Pudjiwati, E.H.; Kuswanto; N. Basuki &

A.N. Sugiharto (2013). Path analysis of

some leaf characters related to downy

mildew resistance in mayze. Agrivita,

35(2), 167–173.

Queenborough, S.A.; M.R. Metz; R.Valencia &

S.J. Wright (2013). Demographic conse-

quences of chromatic leaf defense in

tropical tree communities: Do red young

leaves increase growth and survival.

Annals of Botany, 4, 677–684.

Roelfs, A.P. (1984). Race specificity and methods

of study. pp. 131–164. In: The Cereal

Rusts, Origins, Specificity, Structure

and Physiology (W.R. Bushnell &

A.P. Roelfs, eds). Academic Press,

Orlando, FL.

Schaefer, A.M.; R. McFarland; E.L. Blakely;

L. He; R.G. Whittaker; R.W. Taylor;

P.F. Chinnery & D.M. Turnbull (2008).

Prevalence of mitochondrial DNA

disease in adults. Annual Neurology,

63(1), 35–39.

Sims, D.A. & J.A. Gamon (2002). Relationships

between leaf pigment content and

spectral reflectance across a wide

range of species, leaf structures and

developmental stages. Remote Sensing

of Environment, 81, 337–354.

Susilo, A.W. & I. Anita-Sari (2011). Respons

ketahanan beberapa hibrida kakao

(Theobroma cacao L.) terhadap serangan

penyakit pembuluh kayu (vascular

streak dieback). Pelita Perkebunan,

27, 77–87.

Susilo, A.W.; P. Arisandy; I. Anita-Sari & R. Harimurti

(2016). Relationship analysis between

leaf-stomata characteristics with cocoa

resistance to vascular streak dieback.

Pelita Perkebunan, 32(1), 10–21.

Tellez, P.; E. Rojas & S.V. Bael (2016). Red colora-

tion in young tropical leaves associated

with reduced fungal pathogen damage.

Biotropica. The Journal of the Associa-

tion for Tropical Biology and Conser-

vation, 48(2), 150–153.

Zhang, Q.Y.; Z.L. Li & B.J. Han (2013). Immediate

responses of cyst nematode, soil-borne

pathogens and soybean yield to one-

season crop disturbance after continuous

soybean in northeast China. Interna-

tional Journal of Plant Production,

7, 341–353.

Zhao, Y.R.; X. Li; K.Q. Yu; F. Chneg & Y. He (2016).

Hyperspectral imaging for determining

pigment contents in cucumber leaves

in response to angular leaf spot disease.

Science Reports, 6, 22790.

**0**