Vanilla

Fusarium rot occurs on the roots, stems, fruit, leaves, and shoots of the plant. However, stem and root infections cause the greatest amount of damage, leading to significant yield loss, and hence the disease is often referred to as stem rot, foot rot, or stem and root rot. In Indonesia, Fusarium rot can infect all parts of the plant from nursery to productive plants but the attack on the stem is most harmful. In light of its significance, a greater emphasis on this disease is placed in this chapter.

Historical Review and Distribution

Fusarium rot, attacking the vanilla crop, has been reported since the crop was cultivated commercially in producing countries, including Indonesia, India, Puerto Rico, the Seychelles, Reunion Island, Madagascar, and Polynesia.

A disease on vanilla was first recorded by Zimmermann in Indonesia in 1903 (Tombe, 1994) when infections on stems and leaves were noted. In 1918, symptoms of a root disease were first noticed in vanilla plantations in Mayagüez, Puerto Rico (Alconero and Santiago, 1969). Tucker (1927) reported that the vanilla root rot in Puerto Rico was caused by a soilborne pathogen called Fusarium batatatis var. vanillae Tucker. In 1925, van Hall (in Tucker, 1927) mentioned the occurrence of a root fungus that caused death of vanilla cuttings in Java, Indonesia. However, the earliest reference associating a Fusarium species to vanilla stem rot in Indonesia was by Soetono (1962), who isolated a species of Fusarium from infected vanilla plants and identified it as F. batatatis Wollenweber.

In the Seychelles Islands, where vanilla was formerly an important crop, the same root disease was present according to a report made by Dupont (1921). The disease description coincided quite closely with that in Puerto Rico (Tucker, 1927). Meinecke (in Tucker, 1927) also observed an undescribed Fusarium attacking the tender tips and young pods of vanilla. Averna-Sacca (1930) reported that the same species of Fusarium was isolated from the diseased vanilla growing in Brazil. However, he stated that the Fusarium pathogen only attacked leaves and stems without any mention of its occurrence on the roots.

Lealy (1970) and Owino (2008) reported that root rot of vanilla caused by Fusarium oxysporum (syn. F. batatatis var. vanillae) was the most serious disease in vanilla plantations in Uganda. This and other species of Fusarium, including Fusarium solani have been reported in vanilla plantations in India (Balagopal et al., 1974a, 1974b; Philip, 1980; Anandaraj et al., 2005), Thailand (Ratanachurdchai and Soytong, 2008), Tonga (Stier, 1984), and China (Ruan et al., l998). The pathogen is observed in Reunion Island and Madagascar today, but was probably present as far back as 1871 and 1902, respectively (Bouriquet, 1954).

Symptoms and Disease Development

Symptoms of stem and root rot may appear at any growth stage of the vanilla plant: cuttings, young vines, and mature productive vines in the field. In addition to the stems and roots, the disease attacks other plant parts such as shoots and beans at any time of the year (Tombe et al., 1992a). In Indonesia, most cases of infection occur initially on the stems, followed by roots and shoots, and occasionally on beans and young leaves. Foliar infection is more commonly found on young leaves. During the rainy season infection on shoots is more prevalent than on other parts of the plant, although the damage is not as severe as on the stems.

Under adverse disease development conditions, symptoms appear as black spots on the stems with limited progress and obvious brown margins. On the other hand, under favorable conditions brown to dark brown lesions with less clearly defined margins enlarge and extend very rapidly and spread along the whole stem internode. A chlorotic zone is often observed between the lesion and healthy tissue (Figure 8.1a) (Soetono, 1962; Tombe, 1993a; Anandaraj et al., 2005). Consequently, the infected stem internode constricts, turns brown to dark brown and finally becomes dry and necrotic (Figure 8.1b). However, disease progress appears to be inhibited by nodes along the vine (Figure 8.1c). On the rotted and constricted parts yellowish-orangey-white spore masses are formed, consisting of fungal conidiophores and conidia. When the infected stem is longitudinally cut, necrosis is apparent, as indicated by a brown discoloration, developing from the inner to the peripheral tissue.

On roots the symptoms initially appear in the form of browning, followed by eventual death (Rachmadiono et al., 1982; Tombe, 1993a; Anandaraj et al., 2005). Aerial roots die promptly after entering the soil (Figure 8.1c), resulting in flaccidity and shriveling of the stem and consequently the vine droops. It turns dark brown and decays, and the rot is either soft and watery or somewhat dry, depending on the existing moisture conditions (Alconero, 1968). Fusarium rot of aerial roots is often diffi-cult to distinguish from anthracnose as both have very similar symptoms and the respective pathogens are often coisolated when present in the same farm. Tucker (1927) recorded in some instances that as much as the lower 3 m of a vine may rot away while the upper part remains green and continues its existence. It is, however, important to note that the pathogen can survive within green healthy internodes with no apparent internal or external symptoms.

FIGURE 8.1 Symptoms of Fusarium rot on vanilla vines: (a) dark brown lesion on stem internode with a chlorotic zone; (b) advanced necrotic stem internode and root; (c) numerous aerial roots are produced at the nodes but rot after reaching the soil.

Causal Organism

Tucker (1927) was the first to describe the pathogen of vanilla root rot by detailing the morphology of various spore types and identifying the pathogen as F. batatatis var. vanillae. This name has undergone multiple nomenclatural changes since then. This together with the association of the disease with various plant parts, resulted in the occasional confusion as to the true etiology of the disease. The species name given by Tucker (1927) was on the basis of morphological similarity to Fusarium batatis, the wilt pathogen of sweet potato, but the two pathogens from the respective hosts were not found to be cross-pathogenic, hence, a variety name was used for the vanilla strain. The host specialization of morphologically identical strains of Fusarium species led to the concept of forma specialis (Snyder and Hansen, 1940), whereby many Fusarium species previously named on the basis of host pathogenicity, despite morphological similarity to F. oxysporum Schlechtendahl, were renamed F. oxysporum with different forma specialis epithets according to the hosts. Fusarium oxysporum Schlecht. f. sp. batatis (Wollemw.) Snyder et Hansen was synonymized with F. batatis (Snyder and Hansen, 1940), but it was sometime later that F. batatatis var. vanillae (Tucker) was renamed F. oxysporum Schlecht. f. sp. vanillae (Tucker) Gordon (Gordon, 1965).

F. oxysporum f. sp. vanillae produces various asexual structures: the microconidia, macroconidia, and chlamydospores (Figure 8.2). Mycelia are immersed, sometimes running over the surface of lesions, hyaline, slender; sporodochia on decaying infected part of vanilla; microconidia in false heads and short conidiophores, abundant, single-celled, oval, 4–9 × 2–3.5 μm; macroconidia usually 3- septated, occasionally 1- to 2-septated, rarely 4- to 5-septated, abundant, hyaline, curved, pedicellate, 20–46 × 2.4–8 μm; chlamydospores present, singly or in pairs, thick-walled when old, brown, 6.5–10 μm (mean 7 μm). On potato dextrose agar (PDA) medium the growth of colony is rapid and the white aerial mycelia may become tinged with pale purple.

FIGURE 8.2 Morphology of the F. oxysporum conidia isolated from vanilla on carnation leaf agar: (a) Microconidia produced in short monophialides; (b) Macroconidia produced in aerial mycelium; (c) Chlamydospores formed singly or in short chains; (d) Macroconidia and microconidia.

The general morphological features of the Fusarium isolates from vanilla isolated in Indonesia, Reunion Island, the Comoros, and Central America today are all similar to those of F. oxysporum described by Messiaen and Cassini (1981). They also fit the key descriptions of F. oxysporum (Matuo, 1972). Morphologically, these isolates are also similar to those isolated from vanilla in India (Philip, 1980).

Inoculation tests on five plants with F. oxysporum indicated that the vanilla isolates were pathogenic to vanilla and not pathogenic to melon, cucumber, tomato, and cymbidium, whereas isolates from tomato were nonpathogenic to vanilla (Nurawan, 1990). In a recent study, Xia-Hong (2007) isolated 87 strains of F. oxysporum from vanilla showing stem rot in seven provinces of China. Among these strains, 81 were tested for pathogenicity and only 43 were found pathogenic.

Vegetative compatibility groups (VCG), sometimes called heterokaryon compatibility groups, are a useful tool to identify different genetic groupings in a population of fungi. Each VCG is unique in the sense that the members of one group are not compatible with the members of any other groups. The Indonesian isolates of F. oxysporum f. sp. vanillae have been grouped into two VCGs with another four VCGs represented by single isolates (Tombe et al., 1994b). In Indonesia, isolates within the same VCG were not necessarily restricted to the same region or location (Tombe et al., 1994). These results were similar to those reported in F. oxysporum f. sp. tuberasi from potato (Venter et al., 1992) and Fusarium proliferatum from asparagus (Elmer, 1991).

Studies are currently being conducted on the genetic diversity and population structure of F. oxysporum f. sp. vanillae throughout Indonesia, including reference isolates from various parts of the world. These include the use of PCR-based fingerprinting markers as well as phylogenetic analysis of multiple gene regions. Preliminary results indicate the presence of a relatively high number of haplotypes, some of which form large clonal groups. The 87 strains of F. oxysporum f. sp. vanillae isolated in China (Xia-Hong, 2007) belonged to 12 different VCGs with no correlation between VCG and virulence.

The isolation of nonpathogenic strains of F. oxysporum from vanilla roots led to investigations on the possibility of utilizing some of these strains as biocontrol agents against Fusarium rot of vanilla. Promising results have been observed under experimental conditions, but field efficacy has yet to be verified.

Control Measures

Although several vanilla relatives, such as Vanilla phaeantha, Vanilla aphylla, and Vanilla andamanica, have been shown to be resistant to Fusarium rot (Theis and Jiménez, 1957; Minoo et al., 2008), commercial varieties resistant or tolerant to Fusarium rot have not yet been described. Owing to the fact that Fusarium rot can infect various plant parts at all stages of growth, integrated control measures against this disease are paramount and should be implemented right from the cutting preparation stage, throughout the vegetative and productive phases in the field, until senescence (Hadisutrisno, 1996; Tombe et al., 1997; Anandaraj et al., 2005; Tombe, 2008). Several components of reported control measures among others are the application of benomyl, carbendazim, and mancozeb fungicides (Matsumoto et al., 1992; Anandaraj et al., 2005; Bhai et al., 2006), and biological agents such as Bacillus sp., Pseudomonas fluorescens, and avirulent strains of F. oxysporum (Tombe et al., 1992c, 1997; Hadisutrisno, 1996; Anilkumar, 2004).

Integrated control measures have been developed in Indonesia, which involve the use of (a) pathogen-free and disease-tolerant cuttings inoculated with a nonpatho-genic F. oxysporum strain 10A–M (Figure 8.3) to induce resistance (Bio-FOB cuttings), (b) fungicides, such as benomyl or mancozeb, by dipping cuttings for 20–30 min, (c) biological agents Trichoderma lactae and Bacillus pantothenticus (Figure 8.3), Bacillus firmus and T. lactae or P. fluorescens (Tombe, 2008) premixed with organic material, and (d) botanical fungicide eugenol extracted from clove leaves or clove fruit stalks. Table 8.1 outlines the recommendations for the control of Fusarium rot of vanilla in various scenarios based on an integrated disease management (IDM) approach in Indonesia.

FIGURE 8.3 Biological agents used in the integrated control of Fusarium rot in Indonesia: (a) approximately 3-month-old vanilla cuttings treated with nonpathogenic Fusarium oxysporum strain 10A–M; (b) antagonistic test of B. pantothenticus to F. oxysporum f. sp. vanillae; (c) B. pantothenticus on sucrose peptone agar; (d) Trichoderma lactae on potato dextrose agar.

Although anthracnose of vanilla is generally regarded to be present in most growing regions today, including Indonesia, India, the Comoros, Madagascar, Mexico, Uganda (Bouriquet, 1954; Tombe, 1993b Augstburger et al., 2000; Thomas et al., 2002; Magala, 2008), there are no good records of its distribution and history of its detection and etiology.

Symptoms and Disease Development

Anthracnose symptoms on vanilla appear, especially on older leaves and stems, initially as small gray or black spots, which coalesce or increase in size to become dark brown to black patches or lesions of varying sizes (Taufik and Manohara, 1998) (Figure 8.4a). The disease is also reported to infect young shoots, inflorescence, and beans in India (Anandaraj et al., 2001; Anilkumar, 2004). In advanced disease stages, these lesions may contain the fruiting structures (acervuli), which appear as tiny black dots (Tombe, 1993b). Under humid conditions infective propagules (conidia) are produced and released from the fruiting structures, observed as masses of salmon-pink spore ooze on the surface of the disease tissue (Taufik and Manohara, 1998). These conidia are dispersed by rain splash or carried on various body parts of insects that come into contact with the spore ooze. Infection on the leaves may occur by spore penetrating through stomata or direct penetration. Latent infection occurs when spores penetrate only into a few epidermic cells and does not develop continuously. Only under favorable environmental conditions the fungus will develop and form necrotic spots. Infection on older or senescing leaves does not usually cause significant damage to the crop. The best way to distinguish Fusarium rot from anthra-cnose is to examine the internal tissue where external symptoms are observed. The presence of internal discoloration, in particular within the vascular tissue is indicative of Fusarium rot. Early symptoms of anthracnose are confined to the epidermis or superficial layers while the vascular tissue appears healthy.

FIGURE 8.4 Symptom of (a) Anthracnose on leaf; (b) sclerotium rot (brown water-soaked leaf tissue and white mycelium and sclerotia on soil; (c) Phytophthora stem rot on naturally infected shoot (Inset: symptom after artificial inoculation). (From Andriyani, N. et al. Jurnal Biologi Indonesia, 5, 227–234, 2008. With permission.)

Causal Organism

The causal organism of anthracnose on vanilla is Colletotrichum gloeosporioides Penzig et Saccardo (syn. Colletotrichum vanillae Scalia, C. vanillae Verplancke et Claess, Gloeosporium vanillae Cooke) (Tombe, 1993b; Anilkumar, 2004; Ratanachurdchai and Soytong, 2008). The pathogen is sometimes reported as the teleomorph Glomerella cingulata (Stoneman) Spauld et Schrenk [syn. G. vanillae (Stoneman) Sacc et Traverso], but the sexual stage has never been observed on the plant host. In brief, the morphological description of C. gloeosporioides includes

(1) acervuli disc or cushion-shaped, subepidermal, epiphyllous, 30–345 μm in diameter; conidiophores simple, short, hyaline, setae brown slightly swollen at the base and tapered to the apex on which conidia are occasionally borne 22.5–75 × 2.5–5 μm; and (2) conidia hyaline, unicellular, oval to oblong, 10–17.5 × 3–5 μm.

C. gloeosporioides has quite a wide host range and commonly exists in various kinds of conditions and places. However, the relationship between the vanilla isolates and those from other hosts has yet to be investigated.

Control Measures

Anthracnose on vanilla until now has not been reported to cause great losses to farmers (Taufik and Manohara, 1998; Magala, 2008). However, the disease indirectly affects the overall health of vanilla plants and hence vanilla production. This disease can be controlled using several methods, examples of which are outlined below.

1. Regular pruning of shade trees to control humidity and sunlight. Pruning is preferably carried out at the beginning of the rainy season. Drainage should also be improved at the same time.

2. Eradication of diseased plant parts (by burning for example) to prevent disease spread.

3. Application of fungicides (e.g., benomyl, mancozeb, and carbendazim) especially when disease is recurrent (Taufik and Manohara, 1998; Bhai et al., 2006).

4. Improve plant vigor (e.g., nutrition with organic matter).

This disease generally attacks during the rainy season when the humidity is high and is often found together with Fusarium rot. Sclerotium rot can be a serious problem in the nursery and, whenever associated with Fusarium rot, can result in the death of productive plants in the field (Tombe and Sitepu, 1987; Taufik and Manohara, 1998). The disease is reported in India and Indonesia (Tombe and Sitepu, 1987; Anandaraj et al., 2005), but there are hardly any records on the history and distribution of this disease in other growing regions, probably due to its frequent association and perhaps confusion with the more significant Fusarium rot.

Symptoms and Damage

The disease occurs on the stem base of vanilla, commonly restricted to 5 cm above the soil surface. The diseased stem base becomes water-soaked, brown to dark brown in color, then becoming necrotic and dies. White fluffy mycelium is often found on the infected stem and the surrounding soil surface (Matsumoto, 1993) (Figure 8.4b). Small spherical structures (sclerotia), light brown in color, 0.5–2 mm in diameter, are often observed in association with the mycelium. If the disease occurs in the cutting propagation nursery bed, damage is usually very heavy (Taufik and Manohara, 1998). Sclerotium rot is occasionally found on the vanilla bean, characterized by rotting of bean tips with thick white mat of fungal mycelium, which eventually covers the whole bean. Excess shade, continuous heavy rains, overcrowding of vines, waterlogged conditions, and the presence of the pathogen inoculum in the field are the predisposing factors for bean rot (Anandaraj et al., 2005).

Causal Organism

The causal organism of Sclerotium rot, Sclerotium rolfsii, does not form conidia or any other reproductive structures. However, dormant structures, the sclerotia, are commonly formed (Taufik and Manohara, 1998), which persist in the soil for years. These survival structures are easily spread by rain water splash and run off, contaminated soil, animals and farming equipment (Tombe and Sitepu, 1987), and thus also represent the dispersal and infective propagules of the disease. Under favorable environmental conditions, the sclerotia germinate and form mycelial mats, which colonize host tissue. Characteristic diagnostic symptoms and signs of the disease are the white mycelium around infested plant parts and the light brown sclerotia produced on basal stems and surrounding soil surface. The causal fungus grows well on artifi-cial medium in the laboratory but the teleomorph is never observed. Numerous scle-rotia are formed on the agar surface after 10–14 days. Deriving from the mycelium, these sclerotia are initially white in color and turn pale to darker brown as they mature and form an outer melanized rind.

Control Measures

The following is an outline of recommended control measures for Sclerotium rot of vanilla in Indonesia. Most of these measures are generic for Sclerotium-induced diseases and are hence applicable in other regions.

1. Regular monitoring and early and accurate detection of the disease.

2. Physical removal of diseased tissue, including surrounding plant parts and soil which may contain the sclerotia of the pathogen.

3. Application of biological control agents Trichoderma harzianum, T. lactae, and B. pantothenticus (Tombe, 2007), mixed with organic mulch or compost. The application of T. harzianum mixed in rice grain and sterilized soil, spread on the soil surface of vanilla nursery, was shown to suppress the disease by up to 54% (Kasim and Prayitno, 1993).

4. The use of eugenol as a natural fungicide has also been reported to suppress the disease (Sukamto et al., 1996; Tombe et al., 1997). Ground leaves and flower buds of cloves were spread around the base of vanilla stems or mixed with growing media in the nursery. Eugenol (approx. 10–20% active ingredient) can also be applied by spraying or stem dipping before planting.

5. Application of synthetic fungicides, such as carbendazim, mancozeb, and benomyl, especially in conjunction with the above measures.

This disease has been reported in several vanilla-producing regions in the world, including Indonesia, Madagascar, Puerto Rico, Polynesia, but is generally not as significant as Fusarium rot and anthracnose. However, in Polynesia shoot rot is a serious threat to vanilla production, causing seedling death (Tsao and Mu, 1987). In Indonesia, Phytophthora shoot and bean rot occurs in nurseries and plantations in Bali, Sumatra, and Java (Manohara, 1993), but the disease has not become a serious problem.

Symptoms and Damage

Infected shoots turn light yellowish brown and become completely necrotic in later stages (Figure 8.4c). In Indonesia, disease lesions are usually restricted to the young parts without progressing to the older internodes. The diseased young shoots often die and fall off, after which the older parts are often observed to be further infected with Fusarium rot, which proceeds to cause serious damage (Manohara, 1993). On the shoot, Phytophthora rot is usually characterized by a yellowish brown color on diseased tissue, while Fusarium rot is more typically darker brown (Taufik and Manohara, 1998).

On the bean, Phytophthora rot develops from the tip, forming a water-soaked lesion, which slowly extends toward the pedicel, becoming darker green. The eventual necrosis extends to the whole bunch of beans, sometimes exhibiting abundant external growth of mycelium (Anandaraj et al., 2005). In India and Indonesia, the occurrence of this disease on vanilla plants is heavier during the rainy season (Rachmadiono et al., 1982; Manohara, 1993; Bhai and Thomas, 2000).

Causal Organism

There have been three species of Phytophthora reported to attack vanilla plants, namely Phytophthora palmivora in Polynesia (Tsao and Mu, 1987) and Thailand (Sangchote et al., 2004), Phytophthora capsici in Indonesia (Andriyani et al., 2008), and Phytophthora meadii in India (Bhai and Thomas, 2000).

Morphological characteristics of P. capsici from vanilla in Indonesia include the presence of sporangia, formed sympodially, which vary in shape from ovoid to obpy-riform (Figures 8.5a and b), 35–125 μm long and 17–58 μm wide, and clearly papil-late at the tip. Chlamydospores are also present and formed in the middle of the hypha (Figure 8.5c). Colony morphology varies and vegetative growth (Figure 8.5d) is observed at an optimum temperature range of 25–35°C (Andriyani et al., 2008). The isolates found in Indonesia are heterothallic, belonging to the A1 mating type. The oospores are produced in oogonia with amphygenous antheridia. The three species of Phytophthora reported on vanilla are most easily distinguished from each other based on the sporangial pedicel length: P. capsici with the longest pedicel (10–200 μm) (Figure 8.5a), P. meadii intermediate (10–20 μm) and P. palmivora the shortest (<5 μm) (Figure 8.5b) (Tsao and Mu, 1987; Erwin and Ribeiro, 1996). Further studies on the etiology of this disease are warranted due to the taxonomic confusion of this genus based solely on morphology.

FIGURE 8.5 Morphology of Phytophthora isolates: (a) Sporangial long-branching pattern of P. capsici; (b) sporangial short-branching pattern of P. palmivora; (c) chlamydospore; and (d) mycelium of P. capsici generated on V8 juice agar. (From Andriyani, N. et al. Jurnal Biologi Indonesia, 5, 227–234, 2008. With permission.)

Control Measures

Various recommendations for the control of Phytophthora shoot and bean rot of vanilla include phytosanitation (removal of infected plant parts, implementation of farm hygiene, etc.), control of humidity by pruning shade trees, application of botanical fungicide, such as eugenol (Tombe et al., 1993; Andriyani et al., 2008) and the use of phosphonate compounds, systemic fungicides effective against a wide range of plant diseases caused by many Phytophthora species (Drenth and Guest, 2004).