Variation in susceptibility to Phyllosticta capitalensis-associated leaf disease among inter-specific hybrids, half-sibs and high-yielding clones of Para rubber tree (Hevea)

Abstract

High susceptibility of many commercial clones of Para rubber tree (Hevea brasiliensis) makes disease management very difficult warranting development and use of disease tolerant clones. Hence, hybridization was carried out using high-yielding and susceptible clones of H. brasiliensis viz. RRII 105, RRII 414 and RRII 430 and two disease tolerant species viz. H. spruceana and H. camargoana and two wild Rondônia germplasm accessions viz. RO 380 and RO 2871. The progenies from the breeding are under various stages of evaluation for their growth and rubber yield. During the course of evaluation, it was observed that many progenies were affected by a serious leaf disease in the form of spots which affected only mature leaves. In all leaf samples examined, conidiomata and ascomata were observed from the initial stages of leaf spot symptoms. Based on characteristic morphology of conidiomata and ascomata, the fungus which was consistently detected in the spots was identified as Phyllosticta capitalensis. Occurrence of P. capitalensis in both of its conidiomata and ascomata states with appendaged conidia and appendaged ascospores respectively, in association with the characteristic leaf spots in either H. brasiliensis or its inter-specific hybrids, is a novel report from India or elsewhere. In very few progenies, leaf blight associated with Pestalotiopsis sp., was also observed. In order to find variability in response to the leaf spot disease associated with P. capitalensis, all progenies were assessed for disease incidence and severity. Progenies of crosses viz. RRII 430 x RO 2871, RRII 414 x H. spruceana, RRII 414 x RO 380 and RRII 105 x H. spruceana had more than 80 percent disease incidences in their progenies. Progenies of cross RRII 430 x RO 380 showed minimum disease incidence (I = 61%). Half-sib progenies of a disease tolerant clone Fx 516 showed lesser disease incidences (I = 25-27%) and very low disease index (DI, 0.8-0.9) indicating better tolerance. In addition to the above, few other high-yielding commercial rubber clones were assessed for their susceptibility to P. capitalensis associated leaf spot. In an on-farm trial, two clones viz. RRII 430 and RRII 417 recorded maximum (I = 60%) disease incidence. Clone RRII 422 (I = 10%) followed by RRII 414 (I = 20%) showed minimum disease incidence. Two other clones viz. RRII 105 and RRII 429 showed moderate incidence (I = 50%). P. capitalensis was consistently detected and associated in all of the infected leaf samples from all the host species observed from early stages of disease development. However, more detailed studies are required to ascertain its specific role in disease development in order to develop suitable management strategies.

Full text

Submitted 15 August 2020, Accepted 18 September 2020, Published 19 October 2020
Corresponding Author: Narayanan Chaendaekattu – e-mail – narayanan.chaendaekattu@gmail.com 100
Variation in susceptibility to Phyllosticta capitalensis-associated leaf
disease among inter-specific hybrids, half-sibs and high-yielding
clones of Para rubber tree (Hevea)
Narayanan C* and Reju MJ
Botany Division, Rubber Research Institute of India, Kottayam 686 009, Kerala, India
Narayanan C, Reju MJ 2020 – Variation in susceptibility to Phyllosticta capitalensis-associated leaf
disease among inter-specific hybrids, half-sibs and high-yielding clones of Para rubber tree (Hevea).
Plant Pathology & Quarantine 10(1), 100–110, Doi 10.5943/ppq/10/1/12
Abstract
High susceptibility of many commercial clones of Para rubber tree (Hevea brasiliensis) makes
disease management very difficult warranting development and use of disease tolerant clones.
Hence, hybridization was carried out using high-yielding and susceptible clones of H. brasiliensis
viz. RRII 105, RRII 414 and RRII 430 and two disease tolerant species viz. H. spruceana and H.
camargoana and two wild Rondônia germplasm accessions viz. RO 380 and RO 2871. The
progenies from the breeding are under various stages of evaluation for their growth and rubber
yield. During the course of evaluation, it was observed that many progenies were affected by a
serious leaf disease in the form of spots which affected only mature leaves. In all leaf samples
examined, conidiomata and ascomata were observed from the initial stages of leaf spot symptoms.
Based on characteristic morphology of conidiomata and ascomata, the fungus which was
consistently detected in the spots was identified as Phyllosticta capitalensis. Occurrence of P.
capitalensis in both of its conidiomata and ascomata states with appendaged conidia and
appendaged ascospores respectively, in association with the characteristic leaf spots in either H.
brasiliensis or its inter-specific hybrids, is a novel report from India or elsewhere. In very few
progenies, leaf blight associated with Pestalotiopsis sp., was also observed. In order to find
variability in response to the leaf spot disease associated with P. capitalensis, all progenies were
assessed for disease incidence and severity. Progenies of crosses viz. RRII 430 x RO 2871, RRII
414 x H. spruceana, RRII 414 x RO 380 and RRII 105 x H. spruceana had more than 80 percent
disease incidences in their progenies. Progenies of cross RRII 430 x RO 380 showed minimum
disease incidence (I = 61%). Half-sib progenies of a disease tolerant clone Fx 516 showed lesser
disease incidences (I = 25-27%) and very low disease index (DI, 0.8-0.9) indicating better
tolerance. In addition to the above, few other high-yielding commercial rubber clones were assessed
for their susceptibility to P. capitalensis associated leaf spot. In an on-farm trial, two clones viz.
RRII 430 and RRII 417 recorded maximum (I = 60%) disease incidence. Clone RRII 422 (I = 10%)
followed by RRII 414 (I = 20%) showed minimum disease incidence. Two other clones viz. RRII
105 and RRII 429 showed moderate incidence (I = 50%). P. capitalensis was consistently detected
and associated in all of the infected leaf samples from all the host species observed from early
stages of disease development. However, more detailed studies are required to ascertain its specific
role in disease development in order to develop suitable management strategies.
Plant Pathology & Quarantine 10(1): 100–110 (2020) ISSN 2229-2217
www.ppqjournal.org Article
Doi 10.5943/ppq/10/1/12

101
Key words – Disease resistance breeding – Hevea brasiliensis – Hevea camargoana – Hevea
pauciflora – Hevea spruceana – leaf spot – tolerance – Rondônia – wild germplasm
Introduction
Hevea brasiliensis (Willd. ex. A. de. Juss.) Müell. Arg., belonging to family Euphorbiaceae
(chromosome number, 2n=36), and most commonly known as Para rubber tree, is naturally
distributed in the Amazon forest ranges of South America. The tree yields natural rubber latex
which is a major strategic raw material for more than 40,000 daily-use products including tyres and
hundreds of medical devices and life-saving accessories like latex gloves (Mooibroek & Cornish
2000). The Para rubber tree is susceptible to many leaf and stem diseases caused by phyto-
pathogenic fungi including Phytophthora, Colletotrichum, Corynespora, Oidium and Corticium,
which mostly lead to severe economic loss in rubber yield (Liyanage & Jacob 1992, Jacob 1997,
Jayasinghe 1999, Narayanan & Mydin 2012). Pseudocercospora ulei (Henn.) Hora Junior &
Mizubuti (= Microcyclus ulei (Henn.) Arx.) is a devastating fungal disease causing South American
Leaf Blight (SALB) in Brazil and it is a looming threat to global rubber cultivation (Chee 1990,
Hora Júnior et al. 2014). Recently, serious disease epidemics due to fungal pathogens including
Neofusicoccum, Fusicoccum and Pestalotiopsis reportedly caused huge losses in natural rubber
production in many countries including China, Cameroon, Indonesia, Malaysia and Sri Lanka
(Ngobisa et al. 2013, Liu et al. 2017, Ngobisa 2018). Based on a recent study on H. brasiliensis in
Sri Lanka, few foliar fungal pathogens including Phyllosticta capitalensis Henn. were found to have
host-pathogen association (Herath et al. 2019a, b). However, the above report lacked details on
symptomatology of the foliar diseases and there were no morphological descriptions of the fungi.
Development and use of disease tolerant clones is the only long term strategy for effective and
sustainable management of the diseases (Fernando 1969). In a disease resistant breeding, three
high-yielding clones of H. brasiliensis were hybridized with other disease tolerant Hevea species
viz. H. spruceana (Benth.) Müell. Arg. and H. camargoana Pires and wild Rondônia germplasm
accessions collected from Amazon forests of Brazil. While H. spruceana and H. camargoana have
been used in hybridization for recovering progenies with high level of resistance to SALB and
Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei, the two wild accessions viz. RO 380 and
RO 2871 are putatively tolerant to a few fungal diseases including those caused by Oidium
(Goncalves et al. 1980, Goncalves et al. 1982, Tran et al. 2016, Adifaiz et al. 2017). Open
pollinated progenies were collected from another disease tolerant Brazilian clone Fx 516 which was
earlier used in breeding for resistance to SALB and other major fungal diseases in Sri Lanka and
India (Senanayake & Wijewantha 1968, Fernando & Liyanage 1975, 1980, Narayanan & Mydin
2012). During course of study, a serious leaf disease was found to affect mature leaves of many
progenies. Detailed studies were carried out on the infected leaf samples to identify involvement of
any fungal pathogen. Based on characteristic morphology of conidiomata and ascomata, the fungus
which was consistently detected in the spots was identified as Phyllosticta capitalensis Henn.
Phyllosticta sp., in either ascomatal sexual state (Guignardia heveae Syd. and Syd.) or conidiomatal
asexual state, has been forecasted as an economically important pathogen of H. brasiliensis for
Asian and African regions (Jayasinghe 1999). In subsequent taxonomic treatments, G. heveae has
been grouped under G. mangiferae ‘species complex’ and the name is now considered as synonym
of P. capitalensis (Wulandari et al. 2009). Detailed morphological descriptions of both
conidiomatal as well as ascomatal states of P. capitalensis in association with the characteristic
infectious leaf spots as observed in the present study, has not so far been reported in Hevea. This
paper reports the findings on symptomatology of leaf spot disease, morphology of P. capitalensis
and variation in susceptibility among inter-specific hybrids, half-sib progenies as well as high-
yielding clones of H. brasiliensis. In addition to the above, occurrence of Pestalotiopsis sp. in
association with a minor incidence of leaf blight, is also reported as this fungus is of quarantine
importance in many rubber growing countries.

102
Materials & Methods
Plant material and location
The parental clones were selected from breeding trials located in the Central Experimental
Station of Rubber Research Institute of India (Ranni, Kerala State, India). Through hand pollination
in various cross combinations, high-yielding and susceptible clones of H. brasiliensis viz., RRII
105, RRII 414 and RRII 430 were hybridized with two disease tolerant Hevea species viz. H.
spruceana and H. camargoana and two wild Rondônia germplasm accessions viz. RO 2871 and RO
380, during 2014 to 2016. Two sets of open-pollinated polycross progenies (or half-sibs) were
collected from the disease tolerant inter-specific hybrid clone Fx 516 during 2015 and 2017. Details
of the clones, parentage and country of origin are given in Table 1. All progenies were grown and
maintained in nursery trials (1 x 1 m spacing; C.R.D.) at the experimental station of Rubber
Research Institute of India (RRII; Kottayam, Kerala State, India). RRII is located in Kerala
(09o32’N, 76o 36’E; 73 m above MSL) with a mean annual rainfall of more than 3000 mm. In
addition to above, three more clones viz. RRII 417, RRII 422 and RRII 429, were assessed for the
leaf disease incidence in an on-farm block trial located at Chithalvetty (Punalur, Kerala State,
India). Since the above three clones share same parentage as that of RRII 430 and RRII 414, their
susceptibility to leaf disease gains importance (Table 1). All the above observations were made
during 2019.
Table 1 Details of parental clones and other Hevea species.
Clone (pedigree)/Species
Origin, distribution and habit
Remarks
RRII 105 (Tjir 1* x Gl 1#);
RRII 414, RRII 430
(RRII 105 x RRIC 100);
Hybrids of H. brasiliensis.
India. H. brasiliensis naturally occurs in
South of Amazon river (Brazil, Bolivia,
Ecuador and Peru). Large trees.
High-yielding clones
classified under
Category-I of planting
advisory.
Fx 516 (F 4542 x AVROS 363$)
(Fx, Ford Cross; F 4542 is a
selection of H. benthamiana Müell.
Arg.)
Par ́a (Brazil). H. benthamiana naturally
grows in areas from Colombia to N. Brazil.
Medium to large sized trees.
Tolerant to leaf
diseases caused by
Phytophthora and
Corynespora.
H. camargoana Pires
Brazil. Distributed in Marajo island of
Amazon river delta. Trees of 2-25 m ht.
Variable tolerance to
SALB disease.
H. spruceana (Benth.) Müell. Arg.
Brazil. Naturally occurs in banks of Amazon,
Rio Negro and lower Madeira. Medium sized
tree.
Variable tolerance to
SALB disease.
RO 2871 (Unknown parentage)
(RO, Rondônia)
Brazil. Collected from Rondônia in 1981.
Large trees.
Tolerant to Oidium
leaf disease.
RO 380 (Putative hybrid of H.
pauciflora (Spruce ex Benth.) Müell.
Arg.)
Brazil. Collected from Rondônia in 1981.
Medium to large trees.
Putatively tolerant to
various leaf diseases.
*H. brasiliensis clone, Indonesia; #H. brasiliensis clone, Malaysia; $AVROS, Al-gemene Verneiging Rubber
planters Oostkust Sumatra, Indonesia.
Symptomatology, fungal morphology and percent disease incidence
Progenies and parental clones were affected by a leaf disease in the form of spots of variable
sizes. The disease affected only mature leaves and there was considerable variation in incidence and
severity of the leaf disease among progenies and parental clones. In very few progenies, leaf blight
was observed. Detailed studies were carried out on the symptomatology of the leaf spot and leaf
blight and also on morphology of fungus present in the leaf spots and blights through microscopic
observation using thin sections (15-20 µm). Morphological identification of the leaf spot fungus
was confirmed by the Ajrekar Mycological Herbarium of the National Fungal Culture Collection of
India (Pune, Maharashtra, India), where the infected leaf specimens were deposited with unique
accession number.

103
Percent disease incidence (I = (∑x/N)*100) was estimated as a proportion of diseased plants
where x was number of affected plants and N was total number of plants. Disease severity was
recorded following a modified disease rating scale (Horsfall & Barratt 1945) as follows: 0, spots
absent; 1, spots in less than 25 percent of leaves; 2, spots in 25 to 50 percent of leaves; 3, spots in
51 to 75 percent of leaves; 4, spots in more than 75 percent leaves along with premature leaf fall.
Disease index (DI = ∑(xini)/N) was estimated as the product of incidence and severity of disease
where, where x represented disease rating (0-4), ni represented the number of diseased plants on the
ith grade of the disease scale and n was the total number of diseased plants assessed (Groth et al.
1999). Based on the disease index, the progenies could be rated as tolerant (DI = 0.1-1.0),
moderately tolerant (DI = 1.1-2.0), moderately susceptible (DI = 2.1-3.0) and highly susceptible (DI
= 3.1-4.0). Similarly, observation was also made on disease incidences in the parental clones
located at CES.
Results
Symptomatology of leaf spot disease
Mostly, spots had distinct ash gray, pale or light brown central portion bordered by a narrow
necrotic margin (Fig. 1). The size of the spots was variable according to the development stages but
mature spots were mostly in the range of 8 to 14 mm in diameter. Often, two or more spots
coalesced and gave an appearance similar to that of small blotches thus affecting larger leaf area.
A
B
D
E
F
G
H
I
J
K
L
C
Fig. 1 – Symptoms of leaf spot. A, B Leaf spots in 2-year-old seedling. C Leaf spots in 4-year-old
tree. D Paling of affected leaves. E Infected leaves with typical spots. F Severely infected plant
affected by premature leaf fall. G Leaf in yellowing stage with fungal fruit bodies (arrow).
H Concentric lesions. I Nearby spots colaescing to form small blotches. J, K Spots showing initial
stages of development of fungal fructifications in margin (arrows). L Prolifereation of more
fructifications in the margin of spot. Scale bars: I = 5 mm, J-L = 3 mm. (Copyright: Narayanan
Chaendaekattu)

104
In severe cases, the affected leaves turned greyish brown or pale yellow and withered
prematurely. It was observed that the disease affected only mature leaves. Leaf spots in different
stages of disease development revealed several minute blackish fungal fructifications in the form of
pycnidia and perithecia in various developmental stages (Figs 2, 3). Initially, few isolated
fructifications could be observed in area lining the advancing lesion of the spots (Figs 2, 3).
Subsequently, as the disease progressed into more advance stages, more fructification was
observed.
A
B
C
D
H
I
J
K
L
M
N
O
P
Q
E
F
G
T
R
S
Fig. 2 – Conidiomata of P. capitalensis. A, B Pycnidia in leaf spot. C-E Conidiomata showing
conidial formation (vertical section). F, G Top view and vertical section view, respectively of
ostiole. H-J Conidiogenous cells. K Finer details of ostiole and ruptured leaf tissue.
L-N Conidiogenesis. O-T Conidia. (Note characteristic single apical appendage and
mucilaginous sheath). Scale bars: K = 18 µm, L, M = 20 µm, N = 10 µm, O = 17 µm, P = 20 µm,
Q = 10 µm, R = 5 µm, S = 20 µm, T = 10 µm. (Copyright: Narayanan Chaendaekattu)
Morphological description of the fungus associated with leaf spot
Based on the characteristic morphological features of the anamorphic and teleomorphic stages
detected in the spots, the fungus was identified as Phyllosticta capitalensis Henn. (Barnett & Hunter
1998, Glienke et al. 2011, Wikee et al. 2011, Wikee et al. 2013a, 2013b). The fungus produced
conidiomata and ascomata with characteristic conidiospores and ascospores, respectively (Figs 2-3).

105
Fig. 3 – Ascomata of P. capitalensis. A-D Ascomata in host leaf spots. E-H Vertical sections
showing details of ascomata development. G Mature ascomata with ostiole. H Ascomata showing
ostiole and withest. I, J Asci and ascospores. K-P Ascospores (note the characteristic mucilaginous
appendages of ascospores). Scale bars: F = 15 µm, I = 15 µm, J = 7.5 µm, K = 15 µm, L-M = 10
µm, N = 9 µm, O = 8 µm, P = 10 µm. (Copyright: Narayanan Chaendaekattu)
Phyllosticta capitalensis Henn. Hedwigia 48: 13 (1908) Figs 2-3
Ajrekar Mycological Herbarium (AMH10221)
Conidiomata in leaves pycnidial, epiphyllous, brown or black with a central ostiole. Peridium
thick, lined by brown cells of textura angularis. Conidiogenous cells lining the walls of pycnidium
phialidic, almost cylindrical and hyaline. Conidia ellipsoidal, hyaline, variously guttulate, single
celled, smooth, 8-11×5-6 μm, surrounded by mucilaginous sheath with a single apical appendage,
appendage 4-6 μm long. Ascomata in leaves black, globose to subglobose, unilocular, solitary to
clustered, ostiolate. Pseudoparaphyses absent. Asci bitunicate, fissitunicate, broadly cylindrical to
cylindro-clavate, rounded at the apex, tapering gradually to a long pedicel attached to the basal
peridium, 45-66 × 7-14 μm, 8-spored. Ascospores ellipsoidal, swollen in the centre, single celled,
coarse-guttulate, smooth-walled, 9-16 × 5-8 μm in size, with mucilaginous appendage at each end.
Material examined - in spots of mature leaves of H. brasiliensis, H. brasiliensis x H.
spruceana, H. brasiliensis x H. camargoana, India, Kerala State, Kottayam, farm of Rubber
Research Institute of India, 4 Jan 2020, C. Narayanan, 04012020/Ktym/India/1-5, Ajrekar
Mycological Herbarium, AMH10221.

106
Variation in incidence of Phyllosticta capitalensis-associated leaf spot disease
Details regarding age, number of progenies from each crosses, number of half-sibs, disease
incidence (I) and disease index (DI), are given in Table 2. Based on DI, the families were rated as
tolerant or susceptible. While progenies of RRII 430 x RO 2871, RRII 414 x H. spruceana, RRII
414 x RO 380 and RRII 105 x H. spruceana had more than 80 percent disease incidence, RRII 430
x RO 380 showed minimum disease incidence (61%). Comparatively, progenies of Fx 516 had
more tolerance with low disease incidences (25-27 %). Regarding disease index also, progenies of
Fx 516 showed very low rating (DI, 0.8-0.9) when compared to hybrids (DI, 2.0-2.7).
Table 2 Percent disease incidence (I), and disease index (DI) of progenies
Progeny type
Age (yr.)
n#
I (%)
DI
Clone
n#
I (%)
RRII 105 x RO 380
4
6
69
2.2
RRII 105
143
50
RRII 105 x H. spruceana
4
19
83
2.5
RRII 414
243
20
RRII 414 x RO 380
4
6
84
2.7
RRII 417
397
60
RRII 430 x RO 380
5
95
61
2.0
RRII 422
481
10
RRII 430 x H. camargoana
5
6
74
2.3
RRII 429
240
50
RRII 430 x H. spruceana
5
49
84
2.6
RRII 430
333
60
RRII 430 x RO 2871
5
11
85
2.7
Half-sib of Fx 516
4
91
25
0.8
Half-sib of Fx 516
2
273
27
0.9
#number of plants
All female parental clones of H. brasiliensis viz. RRII 105, RRII 414 and RRII 430, showed
symptoms of leaf spot disease. Most of their hybrid progenies also showed high disease incidence
and disease index (Table 2). Among the male parental clones, H. camargoana and H. spruceana
were asymptomatic without any visible leaf spots. Similarly, wild germplasm male parental clones
(RO 380 and RO 2871) were also asymptomatic, apparently not affected by the disease. Regarding
disease incidence of clones in the on-farm trial, clones RRII 430 and RRII 417 (I = 60%) followed
by RRII 105 and RRII 429 (I = 50%) had high disease incidences indicating less tolerance to the
disease (Table 2). In contrast, clones RRII 422 and RRII 414 showed minimum disease incidences
of 10% and 20% respectively, indicating better tolerance to the disease.
Minor incidence of Pestalotiopsis leaf spot and blight
In very few progenies, ash coloured, irregular, necrotic leaf spot and leaf blights, which were
contrastingly different from those associated with Phyllosticta sp., were observed (Fig. 4).
Microscopic observation of the infected leaves revealed association of Pestalotiopsis sp. with
blights. Conidia were fusoid, ellipsoid, straight or slightly curved (18.1 - 24.9 μm x 5.3 - 6.6 μm)
with four septa and a hyaline, thin-walled and sub-cylindrical apical cell with two to four hyaline
filamentous apical appendages and a basal cell with one hyaline appendage.
Fig. 4 – Pestalotiopsis leaf blight. A Leaf with spots and blight. B Closer view of blight.
C, D Conidia. Scale bars: C = 15 µm, D = 10 µm. (Copyright: Narayanan Chaendaekattu)

107
Discussion
P. heveae Henn. was described in its conidiomatal state from leaves of H. brasiliensis in 1904
(Hennings 1904). G. heveae Syd. and Syd., as the teleomorph state, was described in leaves of H.
brasiliensis from Philippines in 1916 (Sydow & Sydow 1916). G. heveae was later reported from
leaves and seedlings of H. brasiliensis from Selangor, Kuala Lumpur and Peninsular Malaysia
during period 1982 to 1987 (Lee et al. 2012). Later, based on a combined phylogenetic tree
generated through DNA sequence analysis of the internal transcribed spacer region (ITS1, 5.8S,
ITS2), translation elongation factor 1-alpha (TEF1) and actin genes, G. heveae was grouped under
G. mangiferae ‘species complex’, along with several other Guignardia species (Wulandari et al.
2009). In subsequent taxonomic treatments, G. heveae was synonymized with P. capitalensis
(Slippers et al. 2013, Wulandari et al. 2013a, b). Although P. capitalensis was recently reported to
have host-pathogen relationship in H. brasiliensis in Sri Lanka, the report was devoid of
symptomatology of the foliar disease or morphological descriptions of P. capitalensis (Herath et al.
2019a, b). The morphological features of the conidiomata and ascomata states with
characteristically appendaged conidia and ascospores respectively, as described and illustrated in
the present study, were clearly in conformity to those described for P. capitalensis Henn. (= G.
heveae) (Hennings 1908, Wikee et al. 2011, Wikee et al. 2013a, 2013b, Wulandari & To-anun
2014). Based on the existing literature, and based on descriptions from earlier records of P. heveae
or G. heveae reported in H. brasiliensis, occurrence of both conidiomatal and ascomatal states of P.
capitalensis with appendaged conidia and ascospores, in association with the characteristic leaf
spots of H. brasiliensis and its interspecific hybrids with H. spruceana and H. camargoana, has not
so far been reported from India. Also, detailed symptomatology of leaf spots and morphological
descriptions of anamorphic and teleomorphic states P. capitalensis has also not been reported so far
in Hevea.
Our study using several leaf spot specimens collected from different species and clones of
Hevea growing at various locations showed consistent occurrence of P. capitalensis in all samples.
Thus, the study clearly established the association of P. capitalensis in leaf spot symptoms.
However, more specific pathological tests are to be carried out to conclusively establish
pathogenicity and virulence of P. capitalensis in causing infection in different rubber plants under
laboratory conditions. Also, more commercial clones and other species of Hevea should be
examined for occurrence of the above disease in various other rubber growing locations within
India and outside the country, in order to identify potential sources of disease resistance for use in
breeding. Outcome of such study will also help in developing appropriate disease management
strategies including quarantine regulations, particularly in the event of future large-scale infections
in commercial plantations within India and other rubber growing countries. Phyllosticta belong to
fungal family Phyllostictaceae most of which typically infect leaves and fruit, rather than woody
tissue, sometimes causing serious damage (Glienke et al. 2011, Wong et al. 2012). As a plant
pathogenic fungus, Phyllosticta is capable of causing serious damage to the host plants by reducing
photosynthetic ability sometimes even leading to premature leaf or fruit fall (Glienke-Blanco et al.
2002, Baldassari et al. 2008). Several species of Phyllosticta (with or without Guignardia
teleomorph state) have been reported to cause spots on leaves, as well as dark spots and necrotic
lesions on fruits of several plant species (van der Aa & Vanev 2002). As far as potential threat to
global natural rubber plantations, Phyllosticta sp. in either sexual or asexual state has already been
forecasted as an economically important pathogen of H. brasiliensis in Asian and African regions
(Jayasinghe 1999).
Conidial morphology of Pestalotiopsis sp. detected from leaf blight specimens (in the present
study) strongly resembled Pestalotiopsis microspora (Speg.) G.C. Zhao & Nan Li which caused
similar leaf blight of H. brasiliensis in Cameroon (Ngobisa 2018). However, more detailed studies,
including molecular analysis etc., are required to confirm the above. Nevertheless, Pestalotiopsis
sp. is a common plant pathogen prevailing in both tropical and temperate climatic conditions and is
regarded as a non-host specific opportunistic pathogen invading mature leaves of a wide variety of
host plants. Although Pestalotiopsis sp. has been documented as a minor pathogen in Hevea, leaf

108
blight disease caused by P. microspora affected 80% of rubber trees in smallholder's fields in South
West Cameroon causing huge losses (Ngobisa 2018). The pathogen also affected 382,000 hectares
of rubber plantations in Indonesia especially South and North Sumatra, Bangka Belitung as well as
South, Central and West Kalimantan causing 15% reduction in rubber yield (Ngobisa 2018).
Possibilities of more epidemic outbreaks in Sri Lanka due to Pestalotiopsis and Fusicoccum and
precautions to be taken have also been highlighted (http://www.rrisl.gov.lk/annunce_t.php?id=101).
In our study, Pestalotiopsis sp., which was tentatively identified as P. microspora, was observed in
leaf spots and blights of very few progenies and its pathogenic role in development of spots and
associated disease symptoms was also not confirmed. Systematic disease surveys supported by
detailed pathological investigations are required to detect large-scale disease incidences due to
Pestalotiopsis sp., if any, as many high-yielding clones possess unknown levels of tolerance to the
pathogen.
The four high-yielding female parental clones of H. brasiliensis viz. RRII 105, RRII 414 and
RRII 430 and their hybrids were susceptible to leaf spot associated with P. capitalensis. Hence,
more resistant parents are to be identified and used in breeding for developing tolerant clones.
Although other Hevea sp. (H. camargoana and H. spruceana) and wild germplasm (RO 380 and
RO 2871) were asymptomatic and could be inferred as relatively tolerant to the leaf spot disease,
more studies are required to ascertain their tolerance. Clone Fx 516 was found to be highly tolerant
to the leaf spot disease as there was no single incidence of visually detectable leaf spot in any of the
trees. Clone Fx 516 possesses tolerance to many other diseases including Phytophthora abnormal
leaf fall and Corticium pink disease and has been used in breeding for resistance to SALB in Brazil
and Sri Lanka (Senanayaka & Wuewantha 1968, Fernando & Liyanage 1975, 1980). Results from
the present study also indicated that Fx 516 could be a potential parental clone for developing
horizontal resistance to important fungal diseases in Hevea.
Conclusion
Until recently, three genera of fungal pathogens viz. Pestalotiopsis, Neofusicoccum and
Fusicoccum have been mainly reported to cause leaf spot epidemics in Hevea. In the present study,
P. capitalensis was consistently identified from leaf spots which seriously affected intra- and inter-
specific hybrids as well as high-yielding clones of Hevea. Since Phyllosticta sp. has been forecasted
as an economically important pathogen for Asian and African regions, findings from the present
study assume more significance. Detailed follow-up studies are also required to confirm the primary
pathogenic role of P. capitalensis in establishment and spread of leaf spots in order to develop
appropriate disease management strategies. Although Pestalotiopsis sp. was associated with a minor
incidence of leaf blight, its current sporadic occurrence in India merits continuous surveillance as
this fungus has been declared as a quarantine pathogen in many rubber growing countries.
Acknowledgements
Thanks are due to Executive Director, Rubber Board, Kottayam and Director, RRII,
Kottayam, for support and facilities. Assistance of the Curator, National Fungal Culture Collection
of India, Agharkar Research Institute, Pune, Maharashtra, India, in identification and accession of
fungal specimens, is gratefully acknowledged.
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