**5. The search for the causal agent**

#### **5.1 Biotic stress**

#### *5.1.1 Insects*

After the epidemiological explosion of FY in 1986, Embrapa (the Brazilian Agricultural Research Corporation) started conducting studies on insects as a possible vector of the FY causal agent [8]. As the spread of the disease followed the direction of the prevailing winds, while natural barriers - such as roads, rivers, and glades - were not sufficient to prevent it supported this hypothesis [8, 56]. This hypothesis on a possible entomological role in the spread of FY also resided in the fact that this disease has similarities with the lethal yellowing-type disease that affects other palms. This disease that affects several other palms is due to insect-transmitted phytoplasmas [57]. Initially, from inventory obtained in plantations affected by FY in the municipalities of Alvaraes, in the Amazonas State, and Benevides, in the Pará State, the main insects suspected of being responsible for the transmission corresponded to *Persis* sp. and *Myndus crudus* because they are commonly found in oil palm plantations and depend on palm oil for nutrition [15].

Initially, an inventory of insects captured directly on the oil palm plantations located inside and outside areas with FY occurrence was generated. Healthy oil palm plants, isolated in cages made of wood and nylon canvas, received populations of the inventoried insects, and the plants monitored for symptoms appearance. After using almost one million insects in the FY transmission test, no symptomatic plant appeared, and there was no relationship between the affected areas with the collected insect fauna [15, 58]. Additional studies have attempted to establish a link between the insects *Contigucephalus* sp., *Omolicna* sp., and *Myndus crudus* and this disease, but they all gave negative results. Consequently, the authors discarded a Homoptera as the FY vector and suggested new studies on possible very active and rare insect species [8, 56].

Another study attempted to investigate the relationship between the presence of homopterans in the vegetation cover in oil palm plantations and the occurrence of FY [12]. No relation between the vegetation cover and FY occurrence appeared as the disease manifested itself either in an area covered with *Pueraria* spp. as in areas where there were grasses, especially *Brachiaria* spp. [25]. Studies using a series of chemicals in areas where FY occurs - such as insecticides, fungicides, and bactericides - did not reduce the appearance and development of FY [40].

#### *5.1.2 Phytoplasmas*

Phytoplasmas are prokaryotes of the Class Mollicutes that cause diseases in several plant species, including several economically important ones [59]. As biotrophic parasites, they colonize the elements riddled with the phloem and can also be found inside the vectors [60]. These organisms are responsible for Lethal Yellowing (LF), a fatal disease that affects the coconut (*Cocos nucifera* L.) and at least 36 other palm species in the Americas [61, 62].

Insects from the Homoptera order, popularly known as leafhoppers, are the vectors for most phytoplasmas causing disease in plants [63]. Biological characteristics, symptoms, and specificity of the insect vector were the focus of the first studies aiming to associate phytoplasmas with plant diseases [64, 65]. Later, new and more accurate DNA-based techniques started to dominate these studies, leading to the production of specific oligonucleotides for diagnosis [65].

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

Transmission electron microscopy was, for many years, the tool used for the detection and study of the cytological interaction between phytoplasmas and the hosts [66]. Studies using this tool were not successful in associating phytoplasma with FY, been replaced by new molecular techniques for the same purpose [8]. Studies carried out by Brioso et al. [67, 68] using nested-PCR in oil palm plants symptomatic for FY found just a very few samples positives for the presence of phytoplasmas from the SrI and 16SrI groups, which do not allow to associate these phytoplasmas to FY. An attempt to reproduce the disease was carried out by grafting intermediate leaf tips with FY into healthy seedling petioles and, during the period of two years, healthy individuals did not show symptoms characteristic of FY and, thus, the hypothesis proposing phytoplasma as the causal agent was discarded [12].
