*5.1.3 Fungi, bacteria, and nematodes*

In the attempt to establish a causal relationship between plant pathogenic fungi, bacteria, and nematodes with FY, some studies tried to reproduce the symptoms in healthy young and adult oil palm plants inoculated with some of these microorganisms previously isolated from symptomatic plants [69, 70].

A pathogenicity test focused on studying the growth, reproductive and developmental habits of microorganisms, included one-year-old nursery plants with individual inoculations and a mixture of three fungi (*Fusarium* sp., *Pythium* sp., and *Coprinus* sp.) isolated from symptomatic plants; and again, the inoculum was unable to reproduce the disease in healthy oil palm trees [69]. The possibility of mechanical transmission between symptomatic and asymptomatic individuals by some microorganisms was also tested, with no success [69]. The chemical control attempts using fungicides or antibiotics failed to link fungi and bacteria to FY in oil palm [11].

Interestingly, some authors have observed similarities between the disease PC in Colombia and FY in Brazil. Furthermore, the oomycete *Phythophtora palmivora* was reported to be the PC causal agent [7]. The strategy used by Martinez et al. [7] was to remove tissue from oil palm plants exhibiting early symptoms of PC disease to inoculate fruit traps. Once microbial growth was observed in the fruits, tissue was transferred to culture media and pure cultures were obtained. Using the DNA isolated from the pure culture, amplification of the ITS region was performed and sequence analysis showed 99.9% homology to *P. palmivora*. The same study reported pathogenicity tests where sporangia were inoculated into the base of the spear of 150 oil palm nursery plants. After 3 to 4 days, the first symptoms of PC were observed in 85% of the plants [7]. However, full PC symptom development occurred in 15% of inoculated oil palm plants, and depended on environmental conditions. In another experiment, 20 immature spear leaves were inoculated with *P. palmivora,* and 3 days later all tissues were disintegrated, displaying a characteristic odor. Microscopy experiments showed the presence of *P. palmivora* in these tissues, and it was re-isolated using the fruit trap technique.

Nematodes are typically wormlike invertebrates able to live in the soil or inside plant structures such as roots, stems, leaves, and flowers and can cause morphological and developmental changes in their hosts [71]. The hypothesis of a nematode as a causative agent of FY came from observations of FY and the red ring disease caused by the nematode *Bursaphelenchus cocophilus* - in the same area. Ferraz [72] did not observe this nematode in necrotic tissues or young leaves. Some studies found nematodes in the spear leaf rake and young leaves of symptomatic plants and the soil of oil palm plantations with a history of FY but were unable to link it to the appearance of this disease [24, 72].

#### *5.1.4 Viruses and viroids*

Other plant pathogens studied as potential causal agents of FY in oil palm were viruses and viroids. Several methods, including mechanical transmission, grafting, pollen-mediated dispersion, transmission electron microscopy, nested RT-PCR, RCA - rolling circle amplification, and electrophoresis, were used to test the hypothesis of a virus or a viroid as the causal agent of FY, without success [8, 10].

Lin et al. [73] evaluated extracts from plants with and without FY using the polyacrylamide gel electrophoresis technique, and the band patterns generated in both samples did not reveal any apparent difference. The same author also carried out a study to purify virus particles via separation with a fractional density gradient with no success [74]. Kitajima [75] evaluated ultrafine tissues from roots, leaves, and spear leaf of symptomatic and asymptomatic individuals by transmission electron microscopy, but no pathogen could be associated with FY.

Other studies have directed their efforts towards viroids, which are the smallest known phytopathogens, consisting basically of a single-stranded, circular RNA molecule not encapsulated [76, 77]. Beuther et al. [13] searched for viroids and viroid-like RNAs in oil palm plants using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts, with no success in showing a link between this type of pathogen and FY.

#### **5.2 Abiotic stress**

The initial pieces of evidence of a possible abiotic cause for FY came from observations made about the indefinite dissemination pattern in affected areas, with an exponential growth form not observed in the case of biotic stresses [78, 79]. Among the possible abiotic causes linked to the appearance of FY, there are lower and higher amounts of water, high or low temperature, high content of soluble salts in the soil, soil pH unsuitable for oil palm, nutritional deficiencies or excesses, presence of toxic organic compounds and intensity and balance of nutrients [78].

The regions with oil palm plantations and FY occurrence located in the North region of Brazil have soils with patches of quartz sand interspersed with patches of lateritic concretions and are subject to prolonged floodings, 5 to 6 months per year [41]. Thus, studies started aiming to understand the composition of the soil and its influence on FY emergence.

The concentrations of Cu, Fe, Mn, and Zn in the leaves of healthy and symptomatic oil palm plants and resistant interspecific hybrids were determined and found out that their concentrations were below the ideal range, suggesting their involvement in the appearance of FY [80]. Compact soils that stay temporarily saturated by rainfall suffer oxidation by anoxia, making it impossible for plants to absorb Fe [80]. Based on these observations, applications of ferrous sulfate were carried out on plants under different stages of FY, but after 120 days of the experiment, there was no regression of the disease in the evaluated oil palms [80].

The physical properties of the soil from areas with the occurrence of FY revealed that they were naturally well-drained and deep but had a thickening or compacting between the depths of 30 cm and 60 cm, as well as the occurrence of speckles in this depth, which results in soil saturation in the superficial layer during the rainfall season [81]. Bernardes [82] carried out chemical analysis on roots of symptomatic plants, and the results did not allow to pinpoint any element imbalance that could be responsible for FY. Another fact that needs consideration as possibly linked to a potential cause for the disease is the fact that at the moment when the first symptoms appear in the aerial part, the root system is severely impaired, which explains the plants' lack of response to fertilization and other interventions [82].

### *Oil Palm Fatal Yellowing (FY), a Disease with an Elusive Causal Agent DOI: http://dx.doi.org/10.5772/intechopen.98856*

A series of field observations made in the heart of the oil palm production area in Brazil led to new hypotheses for a possible abiotic cause for FY [83]. The main field observations taken into consideration were: a higher occurrence of flooding in oil palm plantations, in comparison to the previous level, observed under native vegetation cover; the layers close to the soil surface without vegetation cover or with oil palm tend to stay close to water saturation for periods much longer than in the native forest; the presence of mottled-iron reduction in the profile of the oil palm plantations, and the redox-potential values (Eh) below −200 mV; and the presence of reduced iron ions on the soil surface in oil palm plantations during periods of intense rain [83].

The new hypotheses were brought together and summarized as: Deficient aeration reduces the potential for oxy-reduction in the soil, causing changes in the ionic composition of the soil solution (reduction of Fe3+ ions; NO3+; Mn3+). The soil solution with a high concentration of reduced ions initially causes damage to the root system (**Figure 3**) predisposing the oil palm plant to physiological disturbances (passive poisoning and attacks of secondary pathogens) whose symptoms are known as FY [84].

To gain insights into the idea of oxygen deficiency (hypoxia) in the origin of FY, a study by Encinas [85] evaluate the influence of land use and temporal variations on the dynamics of nutrients in the solution of soil and water at an oil palm plantation and a nearby area still with primary forest. Another by Muniz [83] compared the changes in water flow at an oil palm plantation and a nearby area still with native vegetation cover and evaluated its effects on iron dynamics and the structure of the soil. These two studies gathered additional shreds of evidence to further support this hypothesis, such as the electrical conductivity increased during a long flooding period (95 days), indicating that ions from the aggregates migrate to the solution; the soil pH increases after the initial flooding period, reaching values close to neutrality, with a subsequent reduction, but above the values found in aerated soil; the soil redox potential decreases during the flooding period, forming a highly reducing environment; the total carbon contained in the macroaggregates reduced

#### **Figure 3.**

*Oil palm plant showing reduction of the root system in hypoxia conditions (A), and soil clouds showing the typical reductimorphic or oximorphic color mottles caused by stagnating soil environment (B). Source: Wenceslau Teixeira.*

after flooding for a period of 11 days; the iron contained in the aggregates of Yellow Latosols with medium texture migrates to the soil solution under flooding conditions; there is a high negative correlation between the iron in the flooding solution and the DMG of the aggregates in the Yellow Latosols, and flooding for a period of 11 days promotes the destabilization of aggregates of Yellow Latosols with medium goethite texture.
