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INDONESIA’S EXPERIENCE OF DEVELOPING GANODERMA TOLERANT/RESISTANT OIL PALM PLANTING MATERIAL

A. Razak Purba*, Umi Setiawati°, Agus Susanto*, Miranti Rahmaningsih°, Yurna Yenni*,

Hernawan Y. Rahmadi* & Stephen P C Nelson°

*Indonesian Oil Palm Research Institute, Jl. Brigjend. Katamso, No. 51, Medan 20158, Indonesia

°Sumatra Bioscience, Bah Lias Research Station, Jl Jend A Yani No 2, PO Box 1154, Medan 20111, Indonesia

ABSTRACT

Ganoderma is a major threat to the South-East Asian oil palm industry. The Ganoderma disease incidence has increased in the past decade. This paper explains the experience of two Indonesian research stations in developing Ganoderma tolerant/resistant planting material to ensure the future sustainability of the Indonesian oil palm industry. The breeding material trials and observations show that there is adequate variation for Ganoderma susceptibility/tolerance for breeders to produce new generations of oil palm planting material, which will be tolerant to Ganoderma. The current trial and nursery test results would suggest that the inheritance of Ganoderma tolerance is generally additive and therefore the performance of crosses can be predicted if parental genotypes have been tested for susceptibility/tolerance to Ganoderma. Most oil palm breeding programmes are heavily reliant on Deli dura as the material used as the female parental palms for seed production. In general this material is highly susceptible to Ganoderma and is therefore essential that either an alternative parental breeding material is identified or the Deli dura material is introgressed with Ganoderma tolerant material. Breeding for Ganoderma tolerant/resistant oil palm planting material is a long term strategy and it is essential that the South-East Asian oil palm industry continues to investigate other alternative prevention and control strategies and does not simply wait for Ganoderma tolerant material to become commercially available.

INTRODUCTION

Oil palm (Elaeis guineensis) was first introduced to Indonesia and South-East Asia

when four African oil palms were planted in the Bogor Botanical Garden, Java, Indonesia in 1848 (Hunger, 1924).

The Indonesian oil palm industry started to develop in the 1950s after the Second World War with a major escalation of oil palm plantings in the mid-1980s. This expansion has continued until today and the total area of oil palm in Indonesia in 2010 was estimated at 7.8 million ha (of which 4.5 million ha is plantations and the balance is smallholder/farmer plantings) (Dirjenbun, 2011). Plantations are usually developed as large scale monocultures with estates being commonly contiguous developments of over 5,000 hectares. Nearly all oil palm plantation companies replant oil palm, after approximately 25 years; to oil palm and no “break” crops are commonly grown between oil palm generations. There has been some conversion of oil palm to rubber and rubber to oil palm. Although the Ganoderma species,

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which infect oil palm, are not pathogenic to rubber Ganoderma species will readily colonise rubber wood as a saprophyte.

Ganoderma infection is able to infect below ground and is thought to usually result from contact between healthy roots and diseased residues left in the ground or the roots of infected palms. It is possible that spores may colonise oil palm tissue on the ground and, thus, lead to oil palm infection when healthy roots make contact with this infected tissue. When there is below ground infection basidiocarps will generally eventually form within 1.5 meters from the ground. Such Ganoderma infection is commonly termed Basal Stem Rot (BSR). Ganoderma spore infection is also possible, although the infection mechanism is not understood. Discreet Ganoderma infection lesions may occur several meters from the ground with the infection not having developed from the ground (Flood, 2002). The only explanation for such lesions is spore infection and these symptoms are described as Upper Stem Rot (USR). Ganoderma is indigenous to Indonesia and is commonly found on dead coconuts as a saprophyte. Ganoderma also plays an important ecological role in Indonesian forests as wood-rotting fungi, which are generally saprophytic although there are some species which are pathogenic.

Ganoderma infection of oil palm was firstly reported in Indonesia in 1931 (Turner, 1981). BSR of oil palm was originally linked with senescence in oil palm (25 to 30 years old) and did not become prevalent until coconuts were replanted with oil palm which resulted in BSR symptoms and palm losses of palms which were 10 – 15 years old. BSR infection of old palms at a low percentage (1-10%) has no real commercial significance in terms of reducing crop yield. This is because there is yield compensation by surrounding healthy palms which are better able to compete for soil nutrients, water and sunlight. It was also emphasized by Turner (1981) that 1% disease incidence was not high enough to reduce oil palm production.

During this past decade, Ganoderma has caused serious economic losses to the Indonesian oil palm industry especially in old plantations or plantations which are in their second, or more, generation of planting. There are direct and indirect losses caused by Ganoderma. Direct losses are because of palm losses while indirect losses are because of a reduction of fresh fruit bunch (FFB) weights because of infection preventing nutrient and water supply to the canopy.

High disease incidence of Ganoderma has been reported in oil palm plantations in Indonesia (Susanto, 2009; Susanto and Huan, 2010). In several old oil palm plantations in Indonesia, the disease incidence has been up to 80% and has caused a major loss in production (Susanto, 2002; Susanto and Sudharto, 2003). At Bah Lias estate, PT PP London Sumatra Indonesia Tbk (Lonsum) in North Sumatra, a 2011 census showed Ganoderma infection of palms less than 6 years old was only 0-1.5%. However, percentage infection increased dramatically in palms more than 16 years old with percentage infection of palms ranging from 13-87% (Virdiana et al., 2011).

During the last decade, Ganoderma infection of immature oil palms has been widely reported particularly in oil palm replantings. Disease incidence tends to increase with each generation of planting. Symptoms appear earlier and with greater severity in the 3rd and 4th generation of oil palm. It has been reported the percentage Ganoderma infection of immature palms in first, second, third, and fourth cycle was 0, 4, 7, and 11% respectively (Susanto et al, 2002). High disease incidence of Ganoderma causes oil palm companies to replant earlier because of a severe decline in yield as a result of low stands per hectare. Therefore oil palm fields are being replanted before they have reached their maximum height (approximately 13 meters) when harvesting of bunches is no longer commercially realistic.

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Decline of fresh fruit bunch (FFB) has been estimated at 0.16 ton/ha for each palm lost. When the stand had declined by 50% the average FFB yield reduction was 35% (Subagio and Foster, 2003).

Ganoderma infection has been reported in almost all oil palm plantations in Indonesia which are no-longer in their first generation of planting. The areas which have the highest losses caused by the disease are North Sumatra, Nanggroe Aceh Darussalam, Lampung and Riau provinces (Susanto, 2009). Therefore, Ganoderma is a destructive pathogen in South East Asia plantation (Semangun, 1990; Treu, 1998; Susanto, 2009), especially in Indonesia and Malaysia (Turner, 1981; Darmono, 1998; Darmono, 2000).

Recently, Ganoderma disease has been partially controlled by cultural practices, biological and chemical techniques. The optimum approach to prevent Ganoderma infection is by breeding for tolerance to Ganoderma and production of tolerant or moderately tolerant commercial palms.

The objective of this paper is to describe the experience of two Indonesian research stations in developing Ganoderma tolerant/resistant planting material. The progress with field and nursery screening trials are explained and the current variation in Ganoderma susceptibility/tolerance. The Ganoderma nursery screening test was developed by CIRAD, Socfindo and Sumatra Bioscience during a collaborate project (Breton et al., 2009).

MATERIALS & METHODS

A. Oil palm germplasm responses toward Ganoderma

To assess the variation of Ganoderma susceptibility/tolerance in IOPRI’s breeding material an observation was conducted to record Ganoderma infection. Typical symptoms of Ganoderma infection were observed, including yellowing fronds followed by necrosis, accumulating spear-shaped fronds, outside layers of fronds hanging, basal stem rot and Ganoderma basidiocarps. The % Ganoderma infection, location of the fields, plant age and generation of plants were recorded. There were 431 crosses observed, including 18 Elaeis oleifera and backcross generations, 143 tenera x tenera/pisifera, 95 dura x dura, and 175 dura x pisifera/tenera crosses.

Ganoderma scoring

Data were analysed using a scoring system for each variable of Ganoderma disease incidence. Variables of disease incidence were field generation, plant age and incidence of Ganoderma disease.

Table 1. Scoring system to measure oil palm tolerance to Ganoderma

Field Generation

Scores for Field

Generation (G)

Plant Age (year)

Scores for Plant Age

(G)

Ganoderma disease

incidence (%)

Scores for Ganoderma

disease incidence

(K) I 1 1 - < 5 1 0 - < 2.5 1 II 2 5 - < 10 2 2.5 - < 5.0 2 III 3 10 - < 15 3 5.0 - < 7.5 3 IV 4 15 - < 20 4 7.5 - < 10 4 20 - < 25 5 10 - < 12.5 5 ≥ 25 6 12.5 - < 15 6 ≥ 15 7

G = Scores for Field Generation + Scores for Plant Age

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K = Scores for Ganoderma disease incidence KR = G – K A plant will have a higher relative tolerance score (KR) when its disease incident score (K) is higher than its overall category score (G). Higher KR means, higher palms tolerance against Ganoderma.

Dura from African TxT crosses observation

IOPRI observed an African TxT 2000 planting at Bah Jambi Estate, PTPN IV, in North Sumatra. There were 42 crosses consisting of 22-45 palms per cross and the observation only assessed Ganoderma infection and individual palm fruit types. The objective was to confirm if the duras from TxT crosses are more tolerant to Ganoderma compared to Deli dura used in IOPRI’s seed gardens. B. Ganoderma field screening trials

Sumatra Bioscience

Genetically diverse DxP breeding progenies were planted in three trials on Lonsum’s Dolok estate in North Sumatra. The soils were alluvial with a high water table - palm losses, as a result of Ganoderma infection, were high with over 50% losses in the previous generation.

The first trial was planted in 2000 and consisted of 127 progenies. The total recorded area was 28.4 Ha. Alpha designs were used with two replicates of 16 palms per plot. The fields were replanted with a 2:1 windrowing system for the old palm stems without soil tillage. A single guard row of a single Deli x AVROS DxP progeny surrounded blocks of four plots. The same trial design and replanting technique was used for the 2001 (with 176 progenies) and 2002 (with 74 progenies planted) field trials.

The objective of these trials was to screen as large a genetically diverse range of progenies as quickly as possible to demonstrate there was significant variation between Sumatra Bioscience (SumBio) progenies for Ganoderma susceptibility/tolerance. It was recognized that the replication was inadequate and the strategy was to replant progenies with the most tolerant progenies after more than six palms per progeny had died. Thus, the most tolerant progenies would be well tested before the end of each trial and exposed to Ganoderma infection.

Four standard breeding crosses were planted in all three trials as references and to allow a combined analysis. Routine individual palm Ganoderma census were conducted every six months. The data recorded at each census included: palm live/dead, the position of basidiocarps (fruiting bodies BSR infection is considered if <1.5 meters from ground and USR if above 1.5m from ground) and foliar symptom scores. A scoring scale (0 to 5) was used where 0 = no symptoms and 5 = severe symptoms). The palms were considered “Ganoderma infected” if the palm had died (after being recorded with Ganoderma symptoms) or alive with the foliar symptoms (score 3-5) and/or basidiocarps had been produced. Turner (1981) mentioned that infected palm symptoms comprise water stress, one-sided mottling of the canopy, flattening of the crown, multiple unopened spears and production of basidiocarps. The number of infected palms out of the total number in a plot, in all three trials at nine years after planting were analysed by Logistic Regression using GenStat 14th edition statistical software. The values generated by this analysis are Predicted Mean % Infection for each progeny (adjusted for trial differences) and the 95% confidence intervals for these means. In 95% of any new trials, the mean is expected to fall within the 95% Confidence Interval range. All significant results are taken as 5% or less.

In addition three trials were planted in three Lonsum North Sumatra estates (Bah Lias, Rambong Sialang, and Gunung Malayu) to test twenty progenies from various breeding materials tested. The trial design was standardized as sixteen replicates with four palms per

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plot. The old palm stems were windrowed (2:1) after felling and there was no soil tillage before planting (Setiawati et al., 2010).

IOPRI Twenty-one high yielding and candidate Ganoderma tolerant DxP progenies were

planted at 2008 in Dolok Sinumbah Estate, PTPN IV, in North Sumatra. Before planting the incidence of Ganoderma infection was mapped and the palm status was recorded as shown in Figure 1 below. The progeny trial design is RCBD with 5 replicates and 12 palms per plot. The observation includes: Ganoderma incidence, vegetative measurement, and eventually bunch analysis and FFB recording.

C. Ganoderma nursery screening trials

Germinated seeds material

A diverse range of genetic backgrounds, from SumBio’s breeding materials, were planted in nursery screening trials. Ganoderma inoculum/disease source

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P A L M N O

30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 58 56 54 52 50 48 46 44 42 40 38 36 34 32 29 27 25 23 21 19 17 15 13 11 9 7 5 3 1 57 55 53 51 49 47 45 43 41 39 37 35 33 31

R O A D

J A L A N

P A R E T

Legends : = Healthy = Ganoderma = Boron = Death

= 434 palms = 92 palms = 3 palms = 1.067 palms

Total = 1.596 palms

Areal = 11.16 ha Population = 143 palms/ha

Figure 1. Ganoderma disease incidence map, prior progeny trial planting.

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Inoculum source preparation was according to the method described by Breton et al. (2005, 2009). Rubber wood blocks (RWBs) were boiled for 5–7 hours and placed in heat tolerant polypropylene bags (2 RWBs/bag) with Potato Agar (PA) medium. The RWBs inside the bag were autoclaved for one hour at 120°C, cooled overnight then inoculated with fragments of mycelium from a PDA fungus culture. The polypropylene bags containing inoculated RWBs were incubated for 12 weeks in the dark at 24–30°C. The Ganoderma isolates used in this study were previously characterized as aggressive. Disease symptoms recording

Disease symptoms were recorded by external observation of seedlings consisted of healthy and infected seedlings. Appearances of dead and living seedlings with leaf symptoms (dried leaves) and/or mycelium and basidiocarp (fruiting bodies) of Ganoderma are categorized as infected seedlings. Observation was conducted at 4, 8, 12, 16, 18 and 20 weeks after germinated seeds were inoculated/planted and at the end of trial (20 weeks) the seedlings were dissected to check for internal mycelia infection within the bole. The severity of internal symptoms was assessed according to a scale established by Breton et al. (2005).

Figure 2. External observation specification consists of healthy seedling (A) and infected seedling (B, C, D) with dry leaves (arrows in B), Ganoderma mycelium and dry leaves (arrows in C) and Ganoderma basidiocarp (fruiting body) and dry leaves (arrows in D)

Design and data analysis 1. Nursery Screening Trials

The design of each nursery screening trial was five replicates of a Randomised Complete Block Design (RCBD) testing 10 – 50 progenies with a plot size of 20 germinated seeds. Logistic regression analysis was used to analyse the variation based on number of infected seedlings at 20 weeks after germinated seeds were inoculated/planted. A significant p-value is taken as <0.05.

2. Isolate x Progeny Trials The design of each isolate x progeny trial was five replicates of Split-Plot Design with different number of plot size (number of germinated seeds). Linear Mixed Model Analysis was used to analyse the variation and interaction based on number of infected seedlings at 20 weeks after germinated seeds were inoculated/planted. A significant p-value is taken as <0.05

A B C D

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RESULTS AND DISCUSSION A. Variation between breeding materials for Ganoderma susceptibility/tolerance

The variation of IOPRI’s breeding materials for Ganoderma susceptibility/tolerance was assessed using the Ganoderma field observation results. With these observations each breeding material’s Ganoderma tolerance was predicted, hence, potential tolerant Deli dura or tenera/pisifera African materials could be selected to produce future generations of tolerant material. The average, maximum and minimum Ganoderma infection in IOPRI breeding materials is shown in Table 2 below.

Table 2. Performance of IOPRI’s breeding population against Ganoderma

Min Incident Max Incident

GD (%) Age GD (%) Age

E.o. SU 6.66 - 57 -

BC BR 23.35 0.00 14 70.00 14

BC SU 46.00 25.00 19 75.00 19

DD DS 41.25 0.00 11 75.98 22

MA 15.50 0.00 11 70.73 13

TI 55.17 - 13 -

MA X DS 55.77 41.54 22 69.12 22

TI X DS 47.10 24.65 13 61.36 13

DS X GB 15.36 0.00 11 66.67 13

TT/P RS 11.51 0.00 6 30.43 10

LM 5.00 0.00 6 15.00 6

YA 7.90 0.00 6 44.44 17

YA X LM 11.22 2.00 17 38.89 17

RS X YA 24.49 0.00 6 55.56 17

RS X LM 15.60 0.00 17 52.78 17

MA X LM 8.96 0.00 16 33.33 17

RS X BJ 15.94 0.00 6 26.09 10

DS X NI 26.09 17.39 10 34.78 10

DP/T DS X DS 34.21 0.00 7 61.11 22

DS X BJ 24.07 12.50 22 33.33 22

DS X LM 9.18 0.00 7 30.56 22

DS X RS 17.73 0.00 7 43.30 19

(DS X GB) X RS 23.17 2.78 16 56.67 19

MA X LM 1.98 0.00 7 9.72 16

MA X MA 32.88 0.00 7 73.33 19

MA X RS 36.88 36.25 20 37.50 20

MA X YA 0.35 0.00 7 1.39 7

TI X RS 29.69 26.39 22 33.33 22

Types

Breeding

Population

Avg GD

(%)

E. o.= Elaeis oleifera BC BR = backcross of E. o. Brazil BC SU = backcross of E. o Suriname. DS = Dolok Sinumbah MA = Marihat TI = Tinjowan RS = RISPA LM = La Mé YA = Yangambi BJ = Bah Jambi NI = Nifor GB = Gunung Bayu

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In general, TxT/P crosses had the lowest disease incidence. It is likely that tolerance to Ganoderma is attributed to the African tenera/pisifera material having a wider gene pool than Deli dura material. So, it is likely that Ganoderma tolerant genes occur in this more genetically diverse material which may not be present in Deli dura. The average disease incidence of each cross is shown in Table 3 below.

Table 3. Ganoderma disease incidence of each cross

No. Cross Disease Incidence (%) 1. Elaeis oleifera Suriname 6.66 c 2. E. o. backcross 36.01 a 3. Dura x Dura 34.02 a 4. Tenera x Tenera/Pisifera 9.99 c 5. Dura x Pisifera/Tenera 17.79 b

Note: different numbers written in the same column that have similar suffix letter mean that they are not different when tested on Duncan’s multiple range test on 5% step

Currently, there are no reports of oil palm materials which are considered totally tolerant to Ganoderma infection. Various reports have shown there is significant variation between genetic materials and crosses for Ganoderma susceptibility/tolerance (Purba et al., 1994; Susanto et.al., 2009). Progeny of African oil palms demonstrate slower rates of Ganoderma infection compared to Deli dura palms (Akbar et al., 1971; Hastarjo and Soebiapradja, 1975). A test of 20 dura x pisifera crosses (Purba et al., 1994) also indicated that tolerant genotypes do exist. E. oleifera x E guineensis crosses and their hybrids in Malaysia (Chung et al., 1994; Sharma and Tan, 1990) support this claim, although this characteristic of being tolerant is not inherited by their backcross generations (Table 3). Franqueiville et al. (2001) also detected variation in Ganoderma susceptibility of breeding materials planted in a field with high Ganoderma disease incidence.

Based on the relative tolerant scores (KR) analysis, 35 candidate crosses with moderate tolerance to Ganoderma infection were selected from 431 tested crosses. The candidates consisted of the following crosses: 1 Elaeis oleifera, 3 DxD, 1 DxP, and 30 TxT/P. Disease infection among these crosses was between 0 – 6.6% (Table 4).

The three selected DxD crosses had a good KR score, and they were progeny of Dolok Sinumbah (DS) materials and recombination of Gunung Bayu and Dolok Sinumbah (GB x DS). The selected TxT/crosses also had a good KR score, and they were recombination of original Yangambi (YA), La Me, SP 540, Dolok Sinumbah (DS), Nifor (NI), Marihat (MA) and Bah Jambi (BJ) materials. The cross between dura DS and pisifera SP 540 was the only DxP cross that had a good KR score. Parents of dura and pisifera were the best dura and pisifera obtained from the second cycle of RRS analysis (Purba, 2008). It has been reported that E. oleifera and its hybrids (Durand-Gasselin, et al., 2005) show tolerance to Ganoderma, and this observation supports this claim because an E. oleifera germplasm originating from Suriname that was already 57 years old had only 6.6% infection (Table 4).

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Table 4. Crosses that are tolerant of Ganoderma

No Crosses Type Genre Age DI (%)

Relative Tolerance. Population

1 Open poll. E.o. 1 57 6.66 4 Suriname 2 MA 1913/96 DD 3 11 0.00 5 GB x DS 3 BA 6736/90 TP 1 17 0.00 4 La Me x SP 540 4 BA 7019/90 TP 1 17 0.00 4 La Me x (MA x RS) 5 BA 6546/90 TT 1 17 2.00 4 (DS x NI) x La Me 6 BA 6965/90 TT 1 17 2.00 4 YA x La Me 7 BA 6120/90 TP 1 17 2.00 4 YA x SP 540 8 BA 8705/90 TP 1 17 0.00 4 YA x (MA x RS) 9 BA 8377/90 TT 1 17 2.00 4 YA 10 BA 5449/90 TT 1 17 2.00 4 YA x (RS x BJ) 11 BA 6125/90 TP 1 17 0.00 4 YA x (RS x BJ) 12 BA 6731/90 TP 1 17 0.00 4 YA x (MA x RS) 13 BA 1713/91 TT 1 16 0.00 4 MA x SP 540 14 BA 1797/91 TT 1 16 0.00 4 MA x La Me 15 BB 1911/91 TT 1 16 0.00 4 YA x (DS x NI) 16 BB 1350/91 TP 1 16 0.00 4 (DS x NI) x (MA x RS) 17 BB 1383/91 TT 1 16 0.00 4 (DS x NI) x SP 540 18 BA 1593/91 TP 1 16 0.00 4 MA x SP 540 19 BB 1514/91 TP 1 16 0.00 4 (DS x NI) x (MA x RS) 20 BA 2200/91 TP 1 16 0.00 4 La Me x DS 21 BB 3064/91 TT 1 16 0.00 4 MA x (DS x NI) 22 BA 1470/91 TT 1 16 0.00 4 MA x SP 540 23 BA 1799/91 TT 1 16 0.00 4 MA x La Me 24 BB 1513/91 TT 1 16 0.00 4 YA x (DS x NI) 25 BB 1220/91 TP 1 16 2.00 4 (DS x NI) x (MA x RS) 26 BA 3392/91 TT 1 16 0.00 4 MA x YA 27 BA 736/91 TT 1 16 0.00 4 (DS x NI) x SP 540 28 BA 966/91 TT 1 16 2.00 4 YA x La Me 29 BA 890/91 TT 1 16 0.00 4 (DS x NI) x (RS x BJ) 30 BB 5253/81 TT 2 24 5.83 4 86GG4 T x 86NN33 T 31 B 5899/81 TT 2 24 1.67 6 86NE33 T x 100TT37 T 32 BA 2825/82 TT 2 24 3.33 5 87S48 T 33 BB 1729/91 DP 1 16 1.39 4 DS x SP 540 34 T 23/85 DD 2 22 0.00 6 DS

35 TI 1/85 DD 2 23 1.01 6 DS DD = dura x dura TT = tenera x tenera TP = tenera x pisifera DP = dura x pisifera Eo = Elaeis oleifera DI = disease incident DS = Dolok Sinumbah MA = Marihat TI = Tinjowan RS = RISPA LM = La Mé YA = Yangambi BJ = Bah Jambi NI = Nifor GB = Gunung Bayu Relative tolerance = scoring systems to measure oil palm tolerance against Ganoderma, explained in Table 1.

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Dura from African TxT crosses observation

The Deli dura gene pool is narrow, hence, the Ganoderma tolerant genes may have

occurred at a low frequency or have been lost during several generations of selection in Indonesia. To introduce the tolerant genes to Deli dura it is possible to cross with African TxT. To investigate this posibility, IOPRI have made field observations of the African TxT crosses, and the results are shown in Table 5 below.

Table 5. Palm health status of different fruit types of African TxT/P crosses

Fruit Types Palm Status Dura Tenera Pisifera Unknown

No. of palms 155 563 351 60 GD (%) 2.58 1.81 3.13 5.00 Dead (%) 8.39 4.52 4.27 26.67 Not infected (%) 89.03 93.67 92.59 68.33

GD = percentage of Ganoderma incidents Dead = percentage of palms death assumed caused by Ganoderma Not Infected = percentage of palms not infected by Ganoderma

It was found that the Ganoderma incidence was up to 10.97% of the duras from TxT

crosses, hence, it was lower than the duras from DxD which reach up to 34.02% (Table 3). However, some Deli dura progenies (Dolok Sinumbah) planted in 1989 and 1988 in 2nd generation plantation are not infected by Ganoderma or only 1.01% infected (Table 4). Nevertheless, from this finding, TxT cross could be used as Ganoderma tolerant sources when crossed with Deli dura.

Cochard et al. (2009) explained that oil palm breeding population used in oil palm seed industries consisted of three groups; Ivorian origin (group I), Beninese, Nigerian, Cameroonian, Congolese and Angolan origins (group II) and lastly Deli origin (group III). With this population structuring Cochard et al. (2009), suggest that in order to benefit from the quality of one group and the complementarity between other groups, and to introgress tolerance to Deli dura, it is necessary to cross Deli (group III) with La Mé population which is known for its tolerance, then re-crossing with group II, to give (Deli × La Mé) × Group II type crosses. These crosses makes it possible to diversify the genetic base of the Deli origin and escape from a blind alley, notably with regard to Ganoderma tolerance, and to effectively exploit the complementarity between groups.

B. Ganoderma field trial results

Sumatra Bioscience Ganoderma Field Screening Trials The ranking and predicted mean infection (adjusted for trial and replicated effect) of

each progeny planted in each of the 2000, 2001 & 2002 field trials at 9 years after planting (YAP) is shown in Figures 3-5. There is significant variation between breeding materials for percentage Ganoderma infection in all three Ganoderma screening field trials. There are still progenies with low percentage Ganoderma infection (0%) and progenies with very high percentage Ganoderma infection (>50%).

From the Ganoderma census data, until 9 YAP, found that BSR (Basal Stem Rot) type is most prevalent in all three field trials. It was confirmed that the most common manifestation of the disease in North Sumatra is basal stem rot (BSR). Almost certainly this

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is due to below ground infection with the infection spreading up from the roots – uprooting the palm and determining whether the infection came from the roots is necessary to confirm this.

There are four progenies from the 2000 trial which have zero Ganoderma infection. The highest percentage infection is 53%. Until 9 YAP, there is only one progeny from 2001 trial that has has zero Ganoderma infection, and the highest incident is similar to 2000 trial (53%). All progenies planted in 2002 trial have Ganoderma infection at 9 YAP.

Figure 3. The predicted mean infection of progenies tested in 2000 Ganoderma field at 9 YAP.

Figure 4. The predicted mean infection of progenies tested in 2001 Ganoderma field at 9 YAP.

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Figure 5. The predicted mean infection of progenies tested in 2002 Ganoderma field at 9 YAP.

Figure 6 below shows that there is variation between progenies for % Ganoderma infection (at 11 YAP) and variations in fresh fruit bunch mean annual (FFBMA-kg/palm/year) at 9 years yield recording. The graph also shows that there is no correlation between Ganoderma tolerance and susceptibility of progenies tested with the FFBMA. It is therefore concluded that it is realistic to select for Ganoderma tolerance without having to sacrifice yield. Another approach to achieve high yielding tolerant material is by introgressing tolerant progenies with high yielding material. It is essential to ensure that the production of Ganoderma tolerant commercial planting material does not result in declining yields because Ganoderma tolerance has been independently selected from yield. Subagio et al. (2003) found that with Deli x AVROS material, the average FFB yield is 27.4 t/ha/yr at 130 palms per hectare but if the palm number then decreased to 100 palms per hectare then the average yield is reduced to only 22.8 t/ha. If the palm number decreases again down to 50% from its original density, then the FFB yield is only about 17.8 t/ha/yr; a FFB yield reduction of about 35%. Losses begin to have a financial effect once the disease affects more than 10% of the stand (Hasan & Turner, 1998).

Figure 6: The correlation between % Infection & FFBMA (kg/palm/year) of progenies tested in 2000 Ganoderma field at 11 YAP & 9 years recording

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IOPRI Ganoderma Field Trial Screening The progeny trial result showed that until 4 years observation the disease incidence has

not appeared in all crosses while palms surrounding plot (Control I – IV) have high disease incidence of Ganoderma relatively (14 palms from 4220 palms). There were some palm losses because of Oryctes rhinoceros infestation. This finding gives breeders confidence that these progenies are Ganoderma tolerant. Nevertheless, further work is still required to verify this finding.

Sumatra Bioscience Ganoderma Screening Multilocation Field Trials

The same breeding progenies were planted in three different locations. At seven years after planting, the analysis shows there are significant differences between location and between progeny on the percentage of Ganoderma infection. There is also a significant interaction between location and progeny. This significant interaction provides a challenge to the breeder to select specific crosses for specific locations. The BLE location has the highest percentage of predicted mean Ganoderma infection compared to RSE and GME location as shown in Figure 7.

Table 6. Summary Logistic Analysis for multilocation field trial

Figure 7: Percentage predicted mean infection in three locations

Fixed Effect p-value of Logistic Analysis

Location <.001

Progeny <.001

Location.Progeny 0.017

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Figure 8: Percentage predicted mean infection of progenies planted in three locations

Figure 8 shows that there is interaction between progeny and location. Some progenies (BL13791, 10894, 10734) have very high % Ganoderma infection at BLE but low at RSE & GME. Progeny BL13789 perform more stable at three different locations. Progeny BL13793 & BL13794 have greater stability at three different locations. These two progenies have similar female pedigrees. C. Ganoderma nursery screening results

Sumatra Bioscience Ganoderma Screening Nursery Trials

1. Relationship between nursery and field results

Figure 9 shows a positive and significant relationship between Ganoderma infection of 15 progenies in nursery and field trials and there is great confidence that the nursery screening tests do provide a reliable screening method to assess progenies for their susceptibility/tolerance to Ganoderma.

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Figure 9. The relationship between % Ganoderma infection of 15 progenies in nursery screening trials (Trials 74 – 253) and a field screening trial (2002 Ganoderma Field Trial)

2. Development of standard crosses

The development of standard crosses (SC GD) used for nursery screening trials was approached by two methods. The first method was progeny selection and the second method was population selection.

Selection of Ganoderma Standard Crosses (SC) from tested progenies

Figure 10 below shows the results from seven first early nursery screening trials. Thirty-two progenies have been selected as Ganoderma standard crosses in three categories: tolerant, medium and susceptible. The selection was not only based on the predicted means of infected seedlings of each progeny, but also on the availability of the parents for continuous crossing.

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Figure 10. Predicted means of infected seedlings (%) by Logistic Regression Analysis from progenies tested in Trials 74 – 88 at 20 weeks after germinated seeds were inoculated (with bars showing 95% confidence interval)

Selection of Ganoderma progenies from different breeding materials

From fourteen early nursery screening trials, a large number of progenies from Deli, AVROS and Deli x AVROS breeding materials were tested. Figure 11 below shows that Deli breeding material is generally more susceptible and AVROS breeding material is more tolerant. The introgression between Deli and AVROS results in a wide range of Ganoderma tolerance/susceptibility where many of them are in the moderately tolerant. Based on these results, some progenies, from these breeding materials and from newer planting materials were selected as Ganoderma Standard Crosses with AVROS progenies being selected for tolerance, Deli for susceptibility and Deli x AVROS for medium SC GD.

Figure 11. Predicted means of infected seedlings (%) by Logistic Regression Analysis from AVROS, Deli x AVROS, and Deli progenies tested in Trials 74 – 117 at 20 weeks after germinated seeds were inoculated (with bars showing 95% confidence interval)

3. Variation between breeding materials and GCA

Population in Ganoderma tolerance

From the first nursery screening trial in 2006 until the last trial in 2011, more than 1,000 progenies have been tested and usually in multiple trials. Some breeding materials have been categorised as AVROS, Binga, Deli, La Me and Other Institution A (OI-A) have been tested in several trials to assess their Ganoderma tolerance/susceptibility (Figure 12). Many progenies from Binga and Deli breeding materials had a higher level of Ganoderma susceptibility, whilst many progenies AVROS, OI-A and La Me were generally more tolerant to Ganoderma. It is more obvious in La Me breeding materials where many of their progenies have lower predicted means for Ganoderma infection.

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Figure 12. Predicted means of infected seedlings (%) by Logistic Regression Analysis from pure breeding materials progenies tested in Trials 74 – 253 at 20 weeks after germinated seeds were inoculated (with bars showing 95% confidence interval)

Genetic Combining Ability (GCA) for Ganoderma tolerance GCA trials provide a statistical method to determine which parents are transmitting

Ganoderma tolerance or whether there are interactions between the two parents (Specific Combining Ability). The first GCA trials were nursery screening trials with the parents were selected from results of field screening trials. Table 7 below shows although the environment effect is greatest, the GCA effects (female and male) contributed more variation than SCA (female x male interactions). The results from this set of progenies would therefore suggest that in general female and male transmit additive partial tolerance (or susceptibility) to their progenies and this should be amenable to selection. Therefore it should be realistic to make breeding progress for partial tolerance to Ganoderma by selecting parents with low GCA estimates for % Ganoderma infection and then crossing them to produce new generations of crosses for further breeding and selection (Rahmaningsih et al., 2010).

Table 7. Percentage of total variation from parents in individual trials at 20 weeks after germinated seeds were inoculated (Random Effect-Only Logistic Analysis)

Random Effects % Total Variation (Trials 183+184)

Trial 12.05% Trial.Replicate 3.37% Female GCA 4.18% Male GCA 2.37% Female.Male SCA 1.75% Environment (Residual) 76.28%

Figure 13 below shows that there is an interaction between female parent groups of tolerance/susceptibility with the male parent groups. Female parent from the susceptible group when crossed with male parent from the male susceptibility group were as expected highly susceptible to Ganoderma infection. However, when male parent from the tolerant group were crossed with female parent from the susceptible group they were more tolerant to Ganoderma infection than when crossed with female parents from the partial tolerant group.

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Figure 13. Predicted means of infected seedlings from four different crossing groups at 20 weeks after germinated seed inoculation (with bars showing 95% confidence interval)

4. Progeny x isolate Ganoderma nursery screening is conducted by only using one isolate. To verify that

there are no progeny x isolate interactions there are routine trials to test different isolates/strains of Ganoderma which were taken from different sites with different progenies. From several trials results of isolate x progeny shown in Table 8, it is concluded that there are no specific interactions between different isolates and different progenies. The significance values of isolate shows Ganoderma isolates have, as expected, significant variation for pathogenicity/aggressiveness.

Table 8. Summary of Linear Mixed Model Analysis of isolate x progeny trials at 20 weeks after germinated seeds were inoculated

Trial No.

Isolate No. Progeny

p-value (Significance)

Isolate Progeny Isolate.Progeny

113 15 5 *** ns Ns

133 13 4 *** * Ns

134 15 2 *** ns Ns

141 15 5 *** ns Ns

204 20 5 *** *** Ns

220 15 4 *** ns Ns

248.1 20 4 * * Ns

248.2 20 4 *** *** Ns

The significance is achieved by LSD test ns is not significant at p = 0.05 * is significant at p < 0.05 ** is significant at p < 0.005 *** is significant at p < 0.001

5. Deli x AVROS From the thirty-six first trials, many progenies from Deli, AVROS and Deli x

AVROS breeding materials have been tested. In general Deli breeding material is more

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susceptible and AVROS breeding material is more tolerant to Ganoderma infection, whilst the introgression of Deli and AVROS results in wide range of Ganoderma tolerance/susceptibility, where many of them are moderate/medium. These findings have remained consistent, as the nursery screening programme has continued. Figure 14 below shows the predicted means of infected seedlings from progenies especially from Deli, AVROS and Deli x AVROS breeding materials.

Figure 14. Predicted means of infected seedlings (%) by Logistic Regression Analysis from Deli, Deli x AVROS and AVROS progenies tested in Trials 74 – 155 at 20 weeks after germinated seeds were inoculated (with bars showing 95% confidence interval)

Reciprocal test for Deli and AVROS breeding materials

The introgression of Deli and AVROS breeding materials results in many progenies in medium range of Ganoderma tolerance/susceptibility. Reciprocal crosses (AVROS x Deli) were made to investigate whether the trait of Ganoderma tolerance/susceptibility is controlled by maternally inherited gene(s). Result from the reciprocal test in Trials 237 and 238 in Figure 15 below shows there is no significant difference between Deli x AVROS and its reciprocal cross.

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Figure 15. Predicted means of infected seedlings (%) by Logistic Regression Analysis from Deli, Deli x AVROS and AVROS progenies tested in Trials 228 and 229 in 20 weeks after germinated seeds were inoculated (with bars showing 95% confidence interval)

CONCLUSIONS

There is great confidence that there is adequate variation for Ganoderma susceptibility/tolerance for breeders to produce new generations of oil palm planting material which will be tolerant to Ganoderma. Although the genetics of Ganoderma tolerance/susceptibility is not well understood there is an increasing understanding of which breeding materials contain more tolerant genotypes and therefore allow breeding progress to be made. The current trial results would suggest that the inheritance of Ganoderma tolerance is generally additive and therefore the performance of crosses can be predicted if parental genotypes have been tested for susceptibility/tolerance to Ganoderma.

Even with nursery screening protocols the breeding approach is long term. The multi-location trials testing the same set of progenies in three different locations show there are progeny x environmental interactions. This is a challenge because these results show that progenies selected for Ganoderma tolerance may not provide the same level of tolerance in different environments.

Most oil palm breeding programmes are heavily reliant on Deli dura as the material used as the female parental palms for seed production. In general this material is highly susceptible to Ganoderma and is therefore essential that either an alternative parental breeding material is identified or the Deli dura material is introgressed with Ganoderma tolerant material. IOPRI are already pursuing the latter strategy and progress is being made.

It is essential that the South-East Asian oil palm industry continues to investigate other alternative prevention and control strategies and does not simply wait for Ganoderma tolerant material to become commercially available. Currently the role of spores in the Ganoderma lifecycle is poorly understood and it is not even known how spores infect oil palm. The percentage of infection which is as a result of spore infection is also unknown. Therefore it is impossible to predict how long it will take for Ganoderma resistance/tolerance to be broken down with the emergence of more aggressive isolates. Since spore infection does occur, thus involving a sexual stage to the life cycle, there is clearly a high risk that tolerance based on a single major gene would put the South-East Asian oil palm industry at high risk to future Ganoderma infection (Setiawati, et al., 2010).

The research on developing cultural practices, biological control (antagonistic fungi etc) and chemical control must continue together in parallel with the breeding programmes to ensure there is an integrated solution to this pathogen. Gaining a greater understanding of the lifecycle of this pathogen and its modes of infection are also essential if effective prevention and control protocols are to be developed.

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