26 individu yang secara genetik merupakan individu-individu yang berbeda Baihaki,
1989. Hipotesis over dominan menjelaskan bahwa vigor hibrida merupakan hasil
penampilan superioritas heterosigositas terhadap homosigositas. Hal in berarti, individu yang berpenampilan superior merupakan individu yang memilki
konstitusi gen heterosigot yang banyak. Genotipe yang heterosigot memiliki tingkat superioritas yang lebih tinggi dibanding dengan genotipe homosigot Fehr,
1987. Menurut Phoelman Sleper 1995 hal tersebut mengandung makna bahwa heterosis terjadi karena adanya interaksi antar gen pada lokus yang sama.
K. Heritabilitas
Nilai heritabilitas merupakan pernyataan kuantitatif peran faktor genetik yang mengukur kemampuan suatu genotip dalam populasi tanaman untuk
mewariskan karakter-karakter yang dimiliki. Pengertian lain menjelaskan bahwa heritabilitas adalah suatu pendugaan yang mengukur sampai sejauh mana
variabilitas penampilan suatu genotip dalam populasi terutama disebabkan oleh peranan faktor genetik. Pemahaman tersebut diperoleh dari pengertian bahwa
pendugaan heritabilitas merupakan perbandingan varian genetik dengan varian fenotip suatu karakter dalam populasi Poehlman Sleper, 1995; Allard, 1960.
Melalui hertabilitas dapat diketahui apakah keragaman yang timbul pada suatu karakter terutama disebabkan oleh faktor genetik atau oleh faktor
lingkungan. Dengan demikian para pemulia tanaman dapat memperlihatkan dari karakter mana yang dapat memberikan respon terhadap suatu usaha perbaikan
yang akan dilakukan. Walaupun heritabilitas merupakan parameter genetik yang memberikan arti besar dalam pemuliaan tanaman, tetapi bukan merupakan suatu
konstanta yang bernilai tetap. Nilai duga heritabilitas adalah parameter yang sangat penting dalam
pemuliaan karena sangat berpengaruh terhadap keefektifan seleksi. Heritabilitas didefinisikan sebagai proporsi total variabilitas yang disebabkan oleh faktor
genetik terhadap variabilitas fenotipik suatu karakter Allard, 1960; Fehr, 1987; Hallauer Miranda, 1988; Crowder, 1993. Heritabilitas tipe ini dikenal sebagai
heritabilitas arti luas broad sense heritabilityh
2 bs
dan diduga sebagai nisbah
27 varians genetik terhadap varians fenotipe.
Varian genetik terdiri atas varian aditif, varians dominan, dan varian interaksi epistasis, sedangkan varian fenotipe adalah varian genetik ditambah
dengan varian lingkungan Falconer, 1989. Heritabilitas didefinisikan sebagai nisbah varian genetik aditif terhadap varian fenotipik. Ini dikenal sebagai
heritabilitas arti sempit narrow sense heritability h
2 ns
yang menggambarkan besar suatu karakter mewaris ke keturunannya Falconer, 1989.
Heritabilitas bukan merupakan besaran yang konstan. Besarnya nilai heritabilitas sangat tergantung pada metode estimasi yang digunakan. Beberapa
metode yang biasa digunakan meliputi: metode pendugaan komponen varian, metode regresi tetua dan keturunan, dan metode pendugaan varian lingkungan
secara tidak langsung Fehr, 1987. Metode lain, yang biasa digunakan untuk menduga heritabilitas arti sempit, adalah dengan menggunakan populasi
backcross Warner, 1952. Klasifikasi tinggi rendahnya heritabilitas suatu karakter ditentukan oleh tinggi rendahnya nilai duga yang diperoleh. Menurut Halloran et
al. 1979, heritabilitas dianggap rendah bila h
2
0,2, sedang bila 0,2 ≤ h
2
≤ 0,5, dan tinggi bila h
2
0,5. Apapun metode yang digunakan, ada beberapa asumsi harus dipenuhi untuk
mendapatkan nilai duga heritabilitas yang akurat. Asumsi tersebut meliputi: 1 tidak ada interaksi non allelik, 2 tidak ada interaksi genetik dengan lingkungan,
3 tidak ada pautan antar gen, dan 4 varian lingkungan pada populasi F2 dan beckcross adalah sama Warner, 1952; Dudly Moll, 1969. Tidak terpenuhinya
asumsi-asumsi tersebut menghasilkan nilai duga heritabilitas yang bias terlalu tinggi atau terlalu rendah.
JUDUL 1. ISOLATION OF INDIGENOUS Phytophthora palmivora FROM
INDONESIA, THEIR MORPHOLOGICAL AND PATHOGENICITY CHARACTERIZATIONS
Abstrak
Patogenisitas isolat P. palmivora dari berbagai sentra produksi kakao di Indonesia belum banyak dievaluasi. Apalagi isolat P. palmivora dapat berubah
dari waktu ke waktu. Sehingga koleksi dan identifikasi keberadaan P. palmivora di lapangan perlu secara periodik dilakukan. Tujuan spesifik penelitian yang
dilakukan adalah: 1 mengkoleksi isolat indigenus Phytophthora palmivora dari sejumlah sentra produksi kakao di Indonesia, 2 mengkarakterisasi isolat
indigenus P. palmivora dari Indonesia menggunakan berbagai karakter morfologi, dan 3 mengevaluasi patogenisitas isolat indigenus P. palmivora terhadap buah
kakao. Isolat indigenus P. palmivora diisolasi dari buah kakao terinfeksi yang berasal dari kebun kakao di 21 kabupaten dan 13 provinsi di Indonesia. Isolat
yang didapat selanjutnya dikarakterisasi morfologi dan patogenisitasnya. Hasil penelitian menunjukkan 24 isolat indigenus P. palmivora telah berhasil diisolasi
dari 13 kabupaten dan 8 provinsi di Indonesia. Isolat indigenus yang didapat mempunyai bentuk spora ellipsoid, globoid, atau ovoid. Sebaliknya, antar isolat
indigenus tidak terdapat perbedaan yang jelas untuk papila dan pediselnya. Meskipun isolat indigenus P. palmivora yang didapat secara morfologis hampir
sama, terdapat perbedaan yang besar dalam tingkat patogenisitasnya terhadap kakao klon GC 7, ICS 60 atau TSH 858. Isolat P. palmivora LBSBR dari Lubuk
Basung, Sumatra Barat diketahui sangat patogenik terhadap ketiga kultivar kakao yang diuji. Sedangkan isolat JkBwi 12 dan KgBwi 8 dari Banyuwangi, Jawa
Timur; PtBdg 7 dari Badung, Bali; SsSpg 36 dan AgSpg135 dari Sopeng,
Sulawesi Selatan, serta PwMnw dari Manokwari, Papua Barat bersifat patogenik
atau sangat patogenik terhadap buah dari tiga klon kakao yang diuji. Kecuali dilakukan pengendalian yang sesuai, isolat indigenous P. palmivora yang bersifat
sangat patogenik atau patogenik dapat berkembang menjadi kendala utama dalam budidaya kakao di Indonesia di masa mendatang.
Kata kunci : kakao, busuk buah, uji buah dipetik, klon GC 7, ICS 60, TSH 858
ISOLATION OF INDIGENOUS Phytophthora palmivora FROM
INDONESIA, THEIR MORPHOLOGICAL AND PATHOGENICITY CHARACTERIZATIONS
Abstract
Pathogenicity of Phytophthora palmivora isolates from various cacao production centers has not been evaluated. Moreover, isolates of this pathogen
may change in time Goodwin, 1997. Therefore, collection and identification of the existing P. palmivora need to be done from time to time. The specific
objectives of this research were: 1 to collect indigenous isolates of P. palmivora from a number of cacao production centers in Indonesia, 2 to characterize the
Indonesian indigenous isolates using various morphological characters, and 3 to evaluate pathogenicity of the indigenous isolates. The indigenous isolates of P.
palmivora were isolated from diseased cacao pod from cacao plantations at 21
districts and 13 provinces in Indonesia. Morphological and pathogenicity characterization were conducted on the identified isolates. Results of the activities
showed that 24 indigenous isolates of P. palmivora were identified from various cacao production centers in 13 districts and 8 provinces in Indonesia. These
isolates produced ellipsoid, globoid, or ovoid spores. On the other hand, they exhibited less apparent differences in their papillae and pedicels. Although these
indigenous isolates of P. palmivora were morphologically similar, they exhibited a diverse pathogenicity against cacao clones GC7, ICS60 and TSH858. The
LBSBR isolate of P. palmivora from Lubuk Basung, West Sumatra were identified as very pathogenic against the three cacao clones tested, while JkBwi
12 and KgBwi 8 isolates from Banyuwangi, East Java; PtBdg 7 isolate from Badung, Bali;
SsSpg 36 and AgSpg1 35 from Sopeng, South Sulawesi, and PwMnw from Manokwari, West Papua were characterized as either pathogenic or
very pathogenic against evaluated pods of the three cacao clones, respectively. Unless properly managed, these pathogenic or very pathogenic indigenous isolates
of P. palmivora might become future major constraints in cacao production in Indonesia.
Keywords: Cacao, black pod, detached pod assay, clones GC 7, ICS 60, TSH
858
Introduction
Black pod disease of cacao is one of the major diseases associated with cacao cultivation in the field Prawirosoemardjo Purwantara, 1992. The cacao
disease caused by Phytophthora palmivora significantly reduced pod and bean yields of cacao. P. palmivora is also capable of infecting stems, young flushes,
and leaves of cacao in the field Purwantara, 1990; Sri-Sukamto, 1985. Yield reduction due to P. palmivora infection in Indonesia was reported up to 45.5
Prawirosoemardjo Purwantara, 1992; Situmorang Soeyatno, 1974 while in other cacao producing countries in the world was between 20 – 30 yearly
Wood Lass 1985. Infection of P. palmivora was one of the major constraints of cacao cultivation in most locations in Indonesia.
In the field, black pod disease is relatively difficult to control because the epidemiology of this disease is very complex Tey, 1991. A number of factor
supporting development of black pod disease in cacao plantation in Indonesia are: 1 the cultivated cacao genotypes are mostly susceptible against black pod
disease, 2 the cacao pods require 5 - 7 months of development before harvesting and they could get infected by black pod disease at any stage of their
development, 3 The relative humidity in Indonesia usually very high all year round, therefore it is favorable for infection and disease development, 4 The
sources of infection are generally always available in the field because of the favorable environment factors and the presence of many alternative hosts in the
field, and 5 the ability of P. palmivora to infect all plant parts of cacao vander Vosen, 1997.
Although P. palmivora could preventively be controlled with fungicides Holderness, 1990, the required cost for controlling this pathogen could reach up
to 40 out of total cost of cacao cultivation Sunaryo Situmorang, 1980. Therefore, availability of alternative methods for controlling P. palmivora is
necessary. A number of antagonistic microbes were able to inhibit development of P. palmivora
Sri-Sukamto et al., 1997, Tondje et al., 2006. However, their effectiveness for controlling black pod disease in cacao needs further evaluation.
Information regarding P. palmivora isolates in Indonesia and other places has been reported Umayah Purwantara, 2006. However, pathogenicity of P.
32 palmivora
isolates from various cacao production centers has not been evaluated. Moreover, isolates of this pathogen may change in time Goodwin, 1997.
Therefore, collection and identification of the existing P. palmivora need to be done from time to time to determine the possible occurrences of new and more
pathogenic isolates in the field. Understanding of the existing P. palmivora isolates infecting cacao and their
characters is needed in order to develop control strategies for the pathogen and to support cacao resistance breeding program for black pod disease Appiah, 2001;
Iwaro et al., 1998; Surujdeo-Maharaj et al., 2001. Therefore, isolation and characterization of pathogenicity of indigenous P. palmivora isolates from various
places in Indonesia need to be conducted. Field isolates of P. palmivora from various cacao production centers in Indonesia may also be used as reference
isolates. Subsequently, they can be used to evaluate resistance of cacao genotypes against infection of P. palmivora.
Research activities supported by the Partnership Cooperation with University in Agricultural Research Project KKP3T have been conducted to
develop effective control strategies for black pod disease in cacao through various approaches, such as studying the indigenous isolates of P. palmivora Sudarsono
et al., 2007. The success of isolating and characterizing indigenous isolates of P.
palmivora is expected to assist the development of effective methods for
controlling black pod disease in cacao and provide the reference isolates of P. palmivora
indigenous Indonesia to cacao breeders. The general objectives of this study were to obtain and characterize
indigenous isolates of P. palmivora – the pathogen causing black pod disease in cacao – from various cacao production centers in Indonesia. The specific
objectives of this research were: 1 to collect indigenous isolates of P. palmivora from a number of cacao production centers in Indonesia, 2 to characterize the
Indonesian indigenous isolates using various morphological characters, and 3 to evaluate pathogenicity of the indigenous isolates.
33
Material and Methods Collection of Diseased Cacao Pods
. Samples of cacao pods infected with black pod disease were collected from cacao plantations at 21 districts and 13
provinces in Indonesia. The provinces and districts were selected because they were known as the center of cacao production in Indonesia or in the process of
developing cacao as one of the major crops in the locations. Within certain province and district, locations where there were concentrations of cacao
plantations were selected for collecting diseased pods. List of locations of diseased cacao pod collection was presented in Table 1. From each location, 2 - 3
cacao pods showing symptoms of black pod infection Fig. 6.a were collected. The sampled cacao pods were either brought back directly or sent through express
mail service to Jember. Subsequently, the diseased pods were used to isolate indigenous P. palmivora in subsequent experiments.
Isolation of Indigenous P. palmivora
. Isolation of P. palmivora from diseased cacao pod was conducted through three steps, such as: 1 baiting step,
2 isolation step, and 3 identification and propagation steps, respectively. For the baiting step, the diseased cacao pods were disinfected using 70 alcohol. A
piece of tissue was cut from diseased pod and used to inoculate a healthy pod of cacao clone GC7 4 months after pollination Fig. 6.b.. To maintain humidity,
the inoculated site of the healthy pods was padded with wet paper towel. Subsequently, the inoculated cacao pods were wrapped with newspaper and
incubated for 5 – 7 days in plastic boxes. The relative humidity in the plastic boxes was maintained at 90.
Once the inoculated pods showed specific symptoms of P. palmivora infection, a piece of tissue from perimeter of diseased and healthy pod was cut
using a scalpel Fig. 6.c. and cultured on a petridish diameter 9 cm containing solid PDA medium Fig. 6.d.. For the isolation step, growing fungal colonies
were purified twice using hyphal tip culture on solid PDA medium. For the identification step, observation was conducted for the ability of the isolated fungal
colonies to produce fungal spore specific for P. palmivora. Fungal colonies capable of producing the specific fungal spore Fig. 6.d. were identified as
isolates of P. palmivora. The identified fungal isolates were propagated on solid PDA medium and stored for further use.
34
Figure 6 Steps of indigenous Phytophthora palmivora isolation from a sample of cacao pod infected with black pod disease in the field and
morphological characters of the isolates. a A sample of cacao pod infected with black pod disease; b Baiting step - inoculation of healthy
pod with a piece of diseased cacao pod; c Occurrences of necrotic symptoms on healthy cacao pod inoculated with a piece of diseased pod
and the tissue in the perimeter of diseased and healthy pod used as inocula for isolation step; d Fungal colonies growing from the
inoculum tissues on solid PDA medium; e Example of typical spore formation from fungal isolate suspected as P. palmivora. Microscopic
observation for the abilities to form those typical spores was conducted to verify the identity of the isolates as P. palmivora; and f Example of
the ovoid O and ellipsoid E types of P. palmivora spore morphologies.
Gambar 6 Tahapan isolasi P. palmivora indigenus dari contoh buah kakao terinfeksi busuk buah dari lapangan dan karakter morfologis isolat. a
Contoh buah kakao terinfeksi; b Baiting step – inokulasi buah kakao sehat dengan potongan contoh buah sakit dari lapangan, c
Kemunculan bercak gejala pada buah sehat yang diinokulasi dengan potongan buah bergejala dari lapangan dan potongan buah pada
perbatasan jaringan yang bergejala dan tidak bergejala hasil baiting yang digunakan sebagai inokulum pada tahapan isolasi; d Koloni
cendawan yang tumbuh dari inokulum pada medium PDA; e Contoh pembentukan spora pada isolat cendawan yang diduga P. palmivora.
Pengamatan mikroskopik untuk kemampuan membentuk spora spesifik tersebut digunakan untuk mengidentifikasi isolat cendawan
yang dievaluasi sebagai isolat P. palmivora; dan f Contoh morfologi spora - ovoid O dan ellipsoid E.
b c
d e
a
f
O E
35
Figure 7 Variation of pathogenicity of indigenous isolates of Phytophthora palmivora
from various cacao production centers in Indonesia based on the width of necrotic symptoms on inoculated cacao pod clones GC7
a,b,c, ICS60 d,e,f, and TSH858 g,h,i. Each cacao pod was inoculated with one indigenous isolate of P. palmivora. Symptoms were
recorded at 3 a,d,g, 5 b,e,h or 7 c,f,i days after cacao pod inoculation with the tested indigenous isolates.
Gambar 7 Variasi patogenisitas isolat P. palmivora indigenus yang berasal
dari sentra produksi kakao di Indonesia berdasarkan lebar bercak pada buah kakao klon GC7 a,b,c, ICS60 d,e,f, dan TSH858 g,h,i. Setiap
buah kakao diinokulasi dengan satu isolat P. palmivora indigenus. Gejala dicatat pada hari ke 3 a,d,g, 5 b,e,h, atau 7 c,f,i sesudah
inokulasi dengan masing-masing isolat.
Morphological Characterization of Indigenous P. palmivora
. Each P. palmivora
isolate identified from previous experiment was grown on a 9-cm petridish containing solid PDA medium. The fungal cultures were incubated for
seven days under dark condition in an incubation room. Temperature in the incubation room was set at 26
o
C day and night. To induce sporulation, mycelia of P. palmivora
grown on solid PDA medium were cooled at 4
o
C for 15 minutes in a refrigerator. Observation for the spore morphology was conducted under
binocular microscope with 100x magnification. In addition, the shape of the fungal spore, the presence of pedicels and papillae were also recorded as part of
a b
c
d e
f
g h
i
36 morphological characters of the P. palmivora isolates.
Pathogenicity Test of Indigenous P. palmivora
. Identified isolates of P. palmivora
were grown on solid PDA medium in Petri dishes, and subsequently were used to inoculate healthy cacao pods in the pathogenicity test. Prior to
inoculation, the pods of cacao clones GC7 susceptible, ICS60 moderately resistant and TSH858 resistant – against infection of P. palmivora Suhendi et
al., 2005 were rinsed in running tap water and damped using tissue towels. The
pods were inoculated with actively growing mycelia on solid PDA medium 0.5x0.5 cm2 and the inoculated site was padded with wetted paper towels. After
inoculation, cacao pods were incubated for 7 days in wooden boxes with 90 relative humidity. To maintain relative humidity, 10 cm thick of foam was laid at
the base of wooden boxes and wetted with sterile water. The boxes were covered with plastic covers and maintained at 28
o
C. Starting at 3 days after inoculation DAP, observations for the occurrences
of necrotic symptoms were conducted daily up to 8 DAP. The observations were conducted on incubation periods, number of pods showing necrotic symptoms,
and average width of necrotic symptoms on the surfaces of cacao pods. Pathogenicity of the isolates was determined based on the diameter of the necrotic
symptoms measured at 8 DAP. The pathogenicity of the isolates was grouped based on criteria developed by Waterhouse 1975, such as: 1 non-pathogenic if
there is no necrotic symptom on the inoculated cacao pods; 2 less pathogenic if the symptom was 25; 3 pathogenic if the symptom was between 25 – 50;
and 4 very pathogenic if the symptom was 50 of the infected cacao pods.
Results and Discussion Isolation of Indigenous
P. palmivora . Isolation of the pathogens from
