Comparative incidence of Ganoderma basal stem rot and upper stem rot disease in oil palm in relation to soil type and geographical distribution

The greatest danger to oil palm sustainability in Indonesia is posed by basal stem rot (BSR) and upper stem rot (USR). However, information on their comparative incidence in relation to soil types and geo-distribution remains scarce. As such, data from annual surveys carried out over 95,000 ha of first-generation oil palms planted in Asian Agri estates was reviewed and analyzed. The highest infection levels occurred in North Sumatra, followed by Riau and Jambi. Likewise, infection levels were highest in peat (24.79 palms/ha), followed by alluvial (13.41 palms/ha), volcanic (7.25 palms/ha), and inland (0.07 palms/ha) soil types. Averaged over three provinces and all four soil types, infection due to USR was more dominant than BSR, comprising 52.3% and 47.7%, respectively. Molecular assays of infected tissue confirmed that both BSR and USR were caused by the genus Ganoderma with the major species being G. boninense. Longitudinal dissections of oil palm trunks infected by USR indicated no infection connectivity with the basal portion of the trunk, unlike BSR, where the infection is through root contact. Transmission through basidiospores is likely to play an essential role in USR epidemiology. Understanding pathogen distribution, species identity, and its transmission epidemiology are prerequisites to control diseases more effectively.


Introduction
Like most perennial crops, the oil palm also suffers infection from some fungal pathogenic diseases.Of the numerous documented to date [1], Ganoderma disease, the causal agent of basal stem rot (BSR) and upper stem rot (USR), is regarded as the significant threat to the oil palm economic stability, especially in Indonesia and Malaysia.In third-generation oil palm replants in North Sumatra, Indonesia, Susanto and Sudharto [2] measured infection levels as high as 75% and hypothesized that infection incidence gradually rises with each succeeding generation of oil palm planting.Likewise, its importance as a major disease in Malaysia was also confirmed when a survey carried out by the Malaysian Palm Oil Board (MPOB) from 2011-2018, revealed that the disease was found to be present in approximately 7.4% and 14.4% of planted acreage of estates and smallholders who participated in the survey, respectively [3].Rao et al. [4] found infection levels as high as 40% in 12-year-old oil palms replanted from both oil palms and coconuts, with potentially infect up to 80% of the palms when the palms are halfway through their economic lifespan [1,5,6].
Although multiple studies have documented a high incidence of BSR in oil palm fields for over 80 years, less is known regarding the status and infection incidence due to USR.The latter's status as a relatively minor disease can be attributed to it being frequently misdiagnosed as BSR due to overlap in the position of infection in the palm trunk [7], or being lumped into the same category as BSR in the 1308 (2024) 012016 IOP Publishing doi:10.1088/1755-1315/1308/1/012016 2 past, most plantations may not have separately recorded BSR and USR during census rounds.In addition, USR infection was initially thought to be caused by Phellinus noxious, rather than by Ganoderma species [1].Since its more recent confirmation as a Ganoderma-inflicted disease, there has been a sharp rise in USR incidence, especially in Sabah and Sarawak, Malaysia [8,9], Papua New Guinea [10], and Indonesia [11,12].
Ganoderma disease, like most oil palm plantations established in South East Asia and Papua New Guinea, is recognized as a key fungal malady impacting first-generation oil palms in Asian Agri estates.High infection rates in volcanic, alluvial, and peat soils, particularly in the province of North Sumatra, Indonesia, had resulted in a considerable loss of palm stands and a quick reduction in production, demanding early replanting [13].Ganoderma, formerly thought to be a disease of oil palms on coastal clays, is now becoming a severe issue even on more free-draining inland, volcanic, and peat soils [13][14][15].
Comprehensive data compiled from annual surveys conducted in 33 estates encompassing four distinct soil types (volcanic, alluvial, inland, peat) and three provinces (North Sumatra, Riau, Jambi) were reviewed and analyzed to gain a better understanding of the status and geographical distribution of BSR and USR disease in first generation oil palms in Asian Agri estates from 2003 to 2013.Furthermore, a predetermined number of tissue samples and Ganoderma fruiting bodies (basidiocarp) were collected from each soil type and province for DNA analysis to confirm the pathogen and species identity.This report presents the findings from the annual surveys and pathogen identity analyses, focusing on soil type and geographical distribution.

Site details
The Ganoderma surveys were conducted from 2003-2013 in 33 selected first-generation oil palm estates in North Sumatra, Riau, and Jambi, encompassing a total area of 40,847, 38,898, and 15,780 hectares, respectively.The first generation Tenera (D x P) oil palms were planted on four distinct soil types (alluvial, volcanic, peat, inland) at a planting density ranging from 132 to 150 palms per hectare.The average rainfall of the surveyed estates ranged from 2,135 mm in North Sumatra to 1,917 mm and 2,480 mm in Riau and Jambi, respectively.
The average age of the oil palms on completing the surveys was 20, 19, and 21 years in North Sumatra, Riau, and Jambi, respectively.Ganoderma disease management in all the estates participating in the survey was per Asian Agri standard operating procedure (SOP), and field maintenance was based on best management practices [13].

Survey details
In each participating estate, every palm in each planting row was inspected for Ganoderma infection at the following frequency per annum (Table 1).Detection of Ganoderma BSR infection was based on external symptoms as described by Turner [1], which included unopened multiple spear leaves, dangling fractured lower and middle-level fronds, and pale coloration of palm canopies.However, according to Asian Agri SOP, the final determination of whether a palm was deemed infected or not was based on the presence or absence of Ganoderma basidiocarps on the palm bole or trunk.
Although late-stage infection symptoms of USR resembled those of BSR, palms in the early stage of infection were often physically healthy and displayed no external symptom just prior to "trunk snapping or breakage."The critical distinction between BSR and USR was that in the latter, the point of infection or presence of fruiting bodies occurred at an elevation higher than one meter from ground level, while BSR infections were below one meter's height, generally at the base of the trunk.In addition, the rotten progress of the palm is also considered.If the rotten tissue starts from the basal stem all the way to one meter high or higher, then this will fall into BSR.Infection higher than one meter but without connectivity with basal rot is categorized as USR [1,7,[17][18][19].During each survey, inspected palms were placed and recorded into either of three categories i.e., healthy palms (category 1), BSR palms (category 2), and USR palms (category 3).

Collection and isolation of BSR and USR causal pathogens
Within Asian Agri estates, 70 samples of infected tissue were obtained randomly from BSR and USRinfected palms from all three provinces and all four soil types.The samples were isolated using a Ganoderma selective medium (GSM), and the pure cultures of Ganoderma isolates obtained were grown on potato dextrose agar (PDA) for subsequent identification, as described by Ariffin and Idris [16].

Molecular identification
The fungal isolation kit's modified mini technique was used to isolate total DNA from all 83 pure isolate cultures.The DNA extraction was done using the Invitrogen DNA isolation kit with minor modification, as done by Haryadi [20].In addition, a multiplex polymerase chain reaction (PCR) was performed following the PCR protocol as described by Boyle et al. [21], with ITS1 and ITS4 as primers.
An Applied Biosystems thermocycler was used for amplification in a 2720 thermal cycler.The amplicons were then sequenced by Bioneer Laboratory in South Korea.The obtained amplicons were further analyzed using DNA sequencing at South Korea's Bioneer Laboratory.The acquired sequences were then evaluated using the Basic Local Alignment Search Tool (BLAST) to discover the most closely similar sequence in the NCBI Genbank database.A phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software, Maximum-likelihood analysis, and 1000 bootstraps to reveal the relationships between the homologous isolates.

Results and discussion
The survey results confirmed the seriousness of the Ganoderma problem faced by Asian Agri oil palm estates on Sumatra island.Over 10 years period, a total of 675,596 palms, or an average of 7.07 palms/ha were recorded to be infected by the disease.This was equivalent to losing 4,791 ha of planted oil palms and would have a significant economic impact on the plantations (Table 2).The survey results also clearly showed the impact of soil type on Ganoderma incidence and severity.Kean and Kerr [22] and Jazuli et al. [23] found that environmental factors, as well as organic matter content, soil fertility, soil moisture, drainage, and the history of fire in native forests, all have a significant impact on soil-borne infections.Of the four soil types assessed in the current study, peat soils recorded the highest infection levels, with an average of 24.79 infected palms/ha.The higher Ganoderma infection levels found in peat compared to the other three soil types can be attributed to several factors.The abundance of organic matter and various plant residues in peat is thought to serve as a substantial source of nutrients.It creates a favorable environment for the rapid build-up of pathogenic Ganoderma inoculum [24,25].Wirianata et al. [26] also reported a faster and higher infection rate of Ganoderma in peat compared to mineral soils, and this was attributed to the factors mentioned earlier.A high water table and a diverse variety of alternative host species in the original peat forests could also be possible causes for the higher infection rates recorded in peat soils.
The higher planting density commercially utilized in peat (150-180 palms/ha) as compared to mineral soils (136-148 palm/ha), could be another contributing factor leading to the higher infection rates reported for peat soils [27,28].Higher planting density results in shorter distances between adjacent palms, facilitating more extensive root contact and a greater potential for infection.In a planting density trial carried out in deep peat, Dolmat [29] confirmed that infection rates at a planting density of 200 palms/ha were 70% and 36% higher than palms planted at 160 palms/ha and 120 palms/ha, respectively.Higher BSR infection is also associated with increased USR incidence in peat soils because more sources of Ganoderma are available in the soils as inoculum, and this opens up opportunities for Ganoderma spores to be carried by wind or insects to the upper trunk of the palm resulting in increased USR incidence.
Out of the three mineral soils, Ganoderma incidence was significantly higher in the clayey and less well-drained alluvium (13.41 palms/ha) as compared to the lighter textured and free draining volcanic (7.25 palms/ha) and inland (0.08 and 0.05 palms/ha in Riau and Jambi, respectively) soils.Higher Ganoderma infection levels in the heavy textured coastal clays as compared to the lighter textured inland soils related to the higher moisture content and poorer drainage in the former, resulting in conditions conducive to infection and development of the pathogen [1,5].However, the interaction between pathogen and its environment is highly complex.The differences in infection levels recorded between soil types could be due to a variety of factors, such as the composition of the original forest species (alternate hosts), which defines the level of indigenous inoculum, soil chemical composition and the presence or absence of suppressive or antagonistic microbes in the soil [30][31][32][33].
Results of the study also highlighted the co-existence of BSR and USR in all surveyed sites, although their composition ratio differed between soil types.In all three mineral soil types (alluvial, volcanic, inland), infection caused by BSR (63.02%) was significantly higher than USR (36.98%).In contrast, infections caused by USR (56.6%) were significantly higher than BSR (43.4%) on peat soils.
The BSR : USR infection ratios recorded in the current study may not apply to all situations and may be site-specific, as the exact factors that influence the ratios remain unknown.Rakib et al. [34] observed no consistent pattern in infection between BSR and USR, while Hasan et al. [35] reported that ratios varied from 1 : 1 to 10 : 1 and, in some areas, the ratios were reversed.As USR infection is thought to be caused by airborne basidiospores [10,36], biotic and abiotic vectors may also be important in their transmission, in addition to specific environmental conditions that are prerequisites for spore germination and fungal mating.Pilotti [10] reported fewer USR infections in closed canopy plantations due to upward flow of basidiospores is restricted, causing them to settle on the ground, resulting in higher BSR infection.On peat where palms are planted at a much higher density than mineral soils, environmental factors such as higher relative humidity, shading intensity, and closer distances between palms would initially be more conducive for USR fungal mating and building up of inoculum.Nevertheless, as the palms age and grow taller, palm leaning and toppling over becomes a common feature of peat plantings, resulting in the creation of more open canopies, spaces, and airflow, facilitating the airborne transmission of basidiospores.USR infections became more dominant where soil conditions were unfavorable for palm growth, particularly in deep peat or in quartzite soil with a sandy pan in the subsoil [1].
About 15 Ganoderma species worldwide were related to BSR [1], while Idris et al. [37] identified four species of Ganoderma associated with BSR in Malaysia, of which three were proven to be pathogenic (G.boninense, G. zonatum, G. miniatocinctum) and one non-pathogenic (G.tornatum).Much less work has been carried out to identify the causal pathogen of USR.Although it was initially thought to be caused by Phellinus noxious [1], more recent studies, have confirmed that the latter disease is also caused by Ganoderma [7,10,35].Pilotti [10] identified G. boninense as the pathogen responsible for stem rot in Papua New Guinea, whilst Rakib et al. [7] found that in Sarawak, Malaysia, both BSR and USR were associated with G. zonatum, G. boninense, and G. miniatocinctum [38], of which G. zonatum was the most dominant pathogen for both stem rots.
Molecular identification of 70 samples collected from Asian Agri estates using multiplex PCR and DNA sequencing revealed two Ganoderma species, G. boninense and G. Miniatocinctum, to be associated with BSR (Table 3).On the other hand, only G. boninense and an unidentified Ganoderma strain were isolated from USR samples (Table 4).Overall, G. boninense was the dominant pathogen, accounting for 93.2% and 96.2% of all samples of BSR and USR-infected tissue, respectively.Similar results were also documented by Susanto et al. [39], who only detected G. boninense in all their samples collected from BSR and USR-infected palms in North Sumatra, Indonesia.Unlike Rakib et al. [7], who recorded G. zonatum being the dominant species in both BSR and USR infections in Sarawak, no cases of the latter species were isolated from Asian Agri plantations.Only a small number of G. miniatocinctum (6.8%) cases were isolated from BSR-infected tissues but none from USR infections.Likewise, a small number of cases (3.8%) of an unidentified Ganoderma species was also isolated from USR palms in the current study.
The molecular analysis conducted in this study further confirmed that the predominant pathogen for BSR Asian Agri Plantations was G. boninense, with the closest identity belonging to G. boninense isolate UPMGB001, which was found in 40.9% of the total isolates (Table 3).The pathogen that is closely related to G. boninense isolate UPMGB001 was also found to be dominant in tissue samples derived from USR palms, comprising 38.5% of the total samples (Table 4).
Although BSR and USR infections were predominantly caused by the same G. boninense isolate, which was closely related to G. boninense isolate UPMGB001, longitudinal dissection of USRinfected palm trunks indicated that infections often occurred at a single point above one meter from ground level.It showed no connectivity with the basal portion of the trunk and, as such, was not a consequence of BSR infection, which occurs typically through the roots or the star cavity of the palm bole.
The DNA sequence data provided accurate and comprehensive information that is highly relevant to endeavors, including evolutionary studies of the origin of organism groups and their geographic distribution, taxonomic classification, and the detection and identification of species across different locations [40][41][42][43].The evolutionary relationship of the pathogens isolated from BSR and USR is presented in Figure 1 and Figure 2. The phylogenetic tree revealed substantial evidence that the BSR and USR causal pathogens isolated from different provinces of North Sumatra, Riau, and Jambi may be closely related among Ganoderma spp.The scaled-down phylogenetic tree had branch lengths that corresponded to the evolutionary distances utilized to infer the phylogenetics.From Figure 1, the dominant strain of G. boninense in BSR that is closely related to G. boninense isolate UPMGB001, grouped close to G. boninense isolate G001, G. boninense isolate GbHap1, and G. boninense strain Gb10 which dominated the abundance of G. boninense in BSR.This suggests that the dominant pathogens may share genetic material influencing their characteristics or aggressiveness.Meanwhile, the dominant strain of G. boninense found in USR, which is closely related to G. boninense isolate UPMG001, was close to G. boninense isolate GB001, the two most closely related strains found in USR samples (Figure 2).Research has shown that populations of G. boninense are primarily made up of distinctive individuals, while some isolates were also discovered to share a single mating allele [44,45].It is clear from this that outcrossing took place over many generations in the native or wild population of Ganoderma, and its impacts may have contributed to the strain diversity.

Conclusion
Developing an appropriate approach for the management of Ganoderma disease requires understanding the pathogen distribution, species identity, and its transmission epidemiology.Of the four soil types assessed in the current study, peat soils in North Sumatera recorded the highest infection levels, with an average of 24.79 infected palms/ha.Results of the study also highlighted the co-existence of BSR and USR in all surveyed sites.However, their composition ratio again differed between soil types and may be site specific.Results from molecular assays confirmed that both BSR and USR were caused by Ganoderma, with the primary species being G. boninense.

Table 1 .
Ganoderma census rotation in Asian Agri

Table 2 .
Comparative incidence of Ganoderma BSR and USR disease encompassing distinct soil types in Sumatra island a Note equivalent to loss ± 4,791 ha

Table 3 .
Pathogen identification of Ganoderma disease isolated from BSR palm

Table 4 .
Pathogen identification of Ganoderma disease isolated from USR palm