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Potential Effects, Biology and Management Options of Coffee Leaf Rust (Hemileia Vastatrix): A Review

Despite coffee plays significant role in social, cultural and national economy of the globe, the coffee industry is potentially at risk due to biotic and biotic stress. Among biotic stress; coffee leaf rust (Hemileia vastatrix) is one of the major disease which has been causing high crop lose world-wide. The breeding programme of coffe leaf rust research center (CIFC) was essentially based on the utilization of HDT as a resistant parent. The main hybrids produced at CIFC with HDT were: HW26 = Caturra Vermelho x HDT 832/1; H 46 = Caturra Vermelho x HDT 832/2; H361 = Villa Sarchi x HDT 832/2; H528 = Catuaí Amarelo x HW26/13; H529 = Caturra Amarelo x H361/3. Ethiopian commercial varieties: Catimor J-19. Catimor J21 and Gesha show partial resistance to the rust. However, in the last few years, some improved commercial varieties are gradually losing their resistance to rust due to the new increase of H. vastatrix virulence races. This shows the importance of re-evaluating existing varieties with the new races and the care needed before the release of new ones. Priority must also be given to the identification of sources of more durable resistance. Ethiopia as the source of origin perhaps for both the host and pathogen plays an important role in science either for breeders or pathologists. Hence, for further advanced investigations on the host and pathogen to improve the durability of coffee resistance to the disease the last ecosystems of rainforest/wild coffee in Ethiopia urgently need to be protected.


COFFEE LEAF RUST (CLR) ( HEMILEIA VASTATRIX )
Coffee leaf rust (CLR) is one of the most important diseases of C. arabica in the world (Kushalappa and Eskes, 1989). Coffee rust has likely been around since Arabica coffee was only growing wild in Africa, but was not 'officially' detected there until the 1870's. Its first recorded impact began in the end of the 19th when a large outbreak in Ceylon (now Sri Lanka) devastated the coffee industry on that small island, ending in the crop being replaced with tea (Abbay, 1876). By the 1960s it had spread throughout Indonesia, and in the 1970s it finally made its way to the Americas, to the Bahia State in Brazil (Correspondent B., 1970;Monaco, 1977). From Brazil, despite the best efforts to quarantine or eradicate the pathogen, it was spread around central and South America over a 15 year period. From what is now known about the viability of spores, it is thought that intercontinental dispersal was possible through wind. Now, it is always around, just in varying degrees. Despite the availability of effective fungicides and resistant varieties for the control coffee leaf rust, it may still cause losses varying between 10 and 40% in different countries . In Brazil, losses have been estimated to be about 30% and an annual loss of about 4500 tons of coffee was estimated in Kenya in the 1960s. The pathogen prefers a temperature range of 20-28 °C needs a leaf wetness period only during spore germination and penetrates with the germination hyphae into the stomata of the host. The fungus tolerates longer seasons without rainfall and spores are wind-borne, only attacking leaves and needs no other host for completing the life cycle. Due to the fact that coffee is a perennial host with green leaves all through the year, the pathogen produces the uredinal, telial, and basidial stages, but only the dycariotic urediospores are responsible for the disease. Coffee grown in lower altitudes is more predisposed to the disease and suffers more attacks. A heavy infestation of leaves not only reduces the assimilation area but also results in a complete defoliation diminishing the next year's crop tremendously. CLR was first reported in Ethiopia in 1934 (Sylvain PG., 1958), but the disease had existed for a long time in other countries without causing epidemics or eradications of certain varieties of C. arabica. The long-term coexistence of coffee and rust coupled with the high genetic diversity of coffee populations and a high level of horizontal resistance might have kept the rust at low levels (Van der Graaff NA., 1981). Other factors such as the low average productivity associated with shade and the existence of biological agents such as the hyperparasite Verticillium lecanii, were also believed to play an important role in maintaining CLR at low levels.

Pathogen Biology
The coffee leaf Rust was first reported by an English explorer on wild Coffea species in the Lake Victoria region of East Africa in 1861. In 1869, the Reverend H. J. Berkeley and his assistant, Mr. Broome, reporting in the Gardeners' Chronicle, described the fungus they found associated with the disease on some dried coffee leaves sent from Ceylon (now Sri Lanka). They gave the name Hemileia vastatrix to the devastating fungus with half-smooth spores ( Figure 1). The Coffee Rust is an obligate parasitic fungus, which means it is a microorganism that must take energy and nutrients from a specific live host (coffee) and reproduces differently than either plants or animals. It belongs to the family of Pucciniaceae in the order of Uredinales of the class Basidiomycetes (Mayne WW., 1932). The genus has unknown pycnidial and aecidial stages and only the dikaryotic uredospores are responsible for the disease development (Kushalappa and Eskes, 1989). The pathogen exists in different physiologic groups.

Potential Effects, Biology and Management Options of Coffee Leaf Rust (Hemileia Vastatrix): A Review
Hemileia vastatrix exists primarily as dikaryotic (having pairs of haploid nuclei that divide in tandem), nutrient-absorbing mycelium ramifying intercellular within the leaves of its coffee host. Clusters of short pedicels bearing dikaryotic urediniospores stick out through the stomata on the undersides of the leaves ( Figure 2). Occasionally under cool, dry conditions toward the end of the season, teliospores are produced among the urediniospores on older, attached leaves. Following karyogamy and meiosis, the teliospores germinate to produce basidia, each of which forms four haploid basidiospores ( Figure 3).

Figure3.
The basidiospores will germinate in vitro, but it is not known what plant, if any, they can infect. It is clear that they do not infect coffee. It is not known whether the basidospores are functional or are simply remnants of an ancestral, long-cycled (up to five different spore stages) rust fungus. No alternate host is necessary; H. vastatrix can survive and reproduce quite nicely by urediniospores alone.Often a hyperparasitic fungus, Verticillium hemileiae, will colonize the coffee rust lesions. Hyperparasites are parasites that parasitize other parasites and are sometimes used as biological control agents. With coffee rust, this hyperparasitism reduces the viability of the urediniospores, but it has very little impact on overall rust development.

Symptoms and Signs
Infections occur on the coffee leaves. The first observable symptoms are small, pale yellow spots on the upper surfaces of the leaves ( Figure 4). As these spots gradually increase in diameter, masses of orange urediniospores (= uredospores) appear on the undersurfaces. The fungus sporulates through the stomata rather than breaking through the epidermis as most rusts do, so it does not form the pustules typical of many rusts . The powdery lesions on the undersides of the leaves can be orange-yellow to red-orange in color, and there is considerable variation from one region to another. While the lesions can develop anywhere on the leaf, they tend to be concentrated around the margins, where dew and rain droplets collect ( Figure 7). The centers of the spots eventually dry and turn brown, while the margins of the lesions continue to expand and produce urediniospores. Early in the season, the first lesions usually appear on the lowermost leaves, and the infection slowly progresses upward in the tree. The infected leaves drop prematurely, leaving long expanses of twigs devoid of leaves ( Figure  8). When a coffee plant does not have the optimal amount of leaf area, it does not have the ability to accumulate adequate energy via photosynthesis and store up the appropriate resources for fruit production. This is why there is generally a loss of yield even the year after rust outbreaks (Avelino and others 2004).

Disease Cycle and Epidemiology
The fungal life cycle is a complex and ingenious one, where organisms asexually produce thousands of tiny spores (reproductive bodies) that can travel in water, rain, or air and remain viable for long distances (Kushalappa and Eskes, 1989).

Figure9.
The disease cycle is a simple one. Urediniospores initiate infections that develop into lesions that produce more urediniospores.

Survival
Hemileia vastatrix survives primarily as mycelium in the living tissues of the host, and since infected leaves drop prematurely (figures 7 and 13); this effectively removes a huge amount of potential inoculum from the epidemic. But a few green leaves always persist through the dry season, and dry urediniospores can survive about 6 weeks, so there is always some viable inoculum to infect the newly formed leaves at the start of the next rainy season.

Spore Dispersal
The urediniospores can be dispersed by both wind and rain. By observing patterns of infection on individual leaves and among leaves within the canopy, it is clear that splashing rain is an important means of local dispersal. The patterns of infection on a regional scale, particularly in those areas where the fungus was newly introduced, have shown that the long-range dispersal is primarily by wind (King'ori and Masaba, 1994). A small, perhaps epidemiologically insignificant amount of urediniospore dispersal is by thrips, flies, wasps, and other insects. Movement across oceans, deserts, and mountain ranges has very likely been caused by human intervention.

Infection
Urediniospores germinate only in the presence of free water (rain or heavy dew) (Nutman and others 1963); high humidity alone is not enough. The whole process of infection requires about 24 to 48 hours of continuous free moisture, so while heavy dew is enough to stimulate urediniospore germination, infection usually occurs only during the rainy season. The seasonal variation in disease incidence is primarily due to variation in rainfall. Where there are two rainy seasons per year, there are two peaks in severity of coffee rust. Temperature also effects rust development. If it is too cold (below 15°C) spores will not germinate and the growth of the fungus will be slowed. Likewise, if it is too warm (more than 35°C) the fungal growth with slow. The most optimal temperature for the growth and proliferation of the rust is between 21-25°C with100percentRH (Nutman et al. 1963).
Light can also change how the fungus affects coffee plants, although there is some mixed evidence around this. Leaves under full sun (high intensity light) have been shown to be more susceptible to rust, and once infected light can alter the growth rate of the fungus. However, very high intensity light has also been shown to slow the fungus growth (Muller et al. 2009). Infection only occurs through stomata on the underside of the leaf.

Fungal infection process
The following properties appear to be characteristics of these fungi: (i) highly developed infection structures; (ii) limited secretory activity, especially of lytic enzymes; (iii) carbohydrate-rich and protein-containing interfacial layers that separate fungal and plant plasma membranes; (iv) haustoria, which are specialized hyphae for nutrient absorption and metabolism; (v) both long-term suppression of host defenses and induction of specific host genes for the establishment of biotrophy (Voegele and Mendgen, 2003).The initiation of the dycariotic phase of H. vastatrix on coffee leaves, as with other rust fungi (Mendgen and Voegele, 2005) involves specific events including appressorium formation over stomata and penetration by inter-and intracellular colonization (Silva et al., 1999a(Silva et al., , 2002. Thus, in susceptible coffee leaves, after urediospore germination and appressorium differentiation over stomata (figure 1C), the fungus penetrates (from 12h after inoculation) (figure1D) forming a penetration hypha (figures 1E) that grows into the sub-stomatal chamber. This hypha produces at the advancing tip two thick lateral branches; each hypha and its branches resemble an anchor (figure 1F). Each lateral branch of the anchor bears a hypha (haustorial mother cell, HMC), the subsidiary cells being the first invaded by haustoria ( figure 1G), whose formation starts around 36h after inoculation. The fungus pursues its growth with formation of more intercellular hyphae, including HMCs, and a large number of haustoria in the cells of the spongy and the palisade parenchyma and even of the upper epidermis.

Sporulation
It takes 10-14 days from infection for new uredinia to develop and urediniospores to be formed. The rust lesions continue to enlarge over a period of 2 to 3 weeks. A single lesion will produce four to six crops of spores, releasing about 300,000 urediniospores over a period of 3 to 5 months. Secondary cycles of infection occur continuously during favorable weather, and the potential for explosive epidemics is enormous.

Fungal Variability
The first race differentiation of H. vastatrix was carried out by Mayne (1932)     The identification proof of the species H. vastatrix by morphological characteristics was assisted by scanning electron microscopic photos of rust sori and urediniospores ( Ritschel A.,2005 ). A typical sorus extruding from a stoma on the lower side of the leave shad15-25 lemon-shapedone-celled urediniospores

Fig12. Urediniosorus and urediniospores of Hemileia vastatrix.
Chala et al. ( 2010 ) suggested that the CLR assessments in the rainforests of Ethiopia revealed its presence in all fields differing in incidence with time (season) and location. A significantly high rust incidence of 31.1% was recorded, for instance, in 2008 at Yayu, followed by Berhane-Kontir (21.4%) and Bonga (7.9%) in forest coffee populations. Rust incidences were consistently highest in Yayu, lower in Berhane-Kontir and lowest in Bonga forests during all seasons. The occurrence of rust in the forest coffee populations varied significantly from season to season. Higher rust incidences were found in January (29.6%) and April (22.7%), while lower incidences were observed in July (13.9%) and October (14.3%). Comparing rust occurrence during the complete period of the surveys from 2003 to 2007 a slight increase of the disease could be observed in the wild coffee population (Fig. 13).

Race types in Ethiopia
There was little emphasis on race-typing of Ethiopian rust samples until the beginning of the 1980s and the 1990s, when the Institute of Biodiversity Conservation (IBC, formerly gene-bank) included coffee in their conservation system. Wondimu et al., (1987) observed that race III was the most frequent in forest coffee and race II in other areas. Other races were I, X and XV. In 2005 the first race-typing of CLR collections of indigenous coffee was carried out at the Centre of Coffee Leaf Rust Research (CIFC) in Oeiras, Portugal using their differentials (Varzea, personal communication). In this study the race specification identified race II at Berhane-Kontir and race III and X in Bonga with corresponding virulence genes v 1, 4 and 5 (Adugna G . et al, 2008).

INHERITANCE OF RESISTANCE
Based on quantitative and Mendelian genetic studies (Bettencourt AJ, 1988), the occurrence of at least nine dominant resistance (R) genes (SH1-SH9), singly or associated in Coffea spp., and a similar number of fungal virulence genes virulence (v1-v9), has been inferred. Major genes SH1, SH2, SH4 and SH5 were found in pure Arabicas from Ethiopia origin, the gene SH3 is considered to derive from C. liberica, and SH6, SH7, SH8 and SH9 were only found in Hibrido de Timor (HDT) (C. arabica x C. canephora) derivatives, therefore supposedly coming from the Robusta side of the hybrid (Bettencourt and Rodrigues Jr., 1988). Besides these SH genes it is likely that other major and minor genes might condition the coffee-rust interactions (Bettencourt and Rodrigues Jr., 1988).
It is thus commonly accepted that the outcome of coffee/rust interactions, whether the plant resists pathogen attack (incompatibility) or develops disease (compatibility), relies on the gene-for-gene model (Flor, 1942) which has been recently amended (Jones JD and Dangl JL., 2006). Once delivered into coffee cells, H. vastatrix effector proteins, and the intracellular perturbations that they trigger, are supposed to be perceived by specific R-proteins. The recognition step promotes the launching of signaling defense pathway(s) and subsequent resistance. Alternatively, virulent rust races are believed to secrete effectors that escape or even counteract the host surveillance system, which allow for the high jacking of coffee cell metabolism and tissue colonization (Fernandez D, 2011).
The coffee genotypes are classified based on complete resistance or susceptibility to several rust races. Group A, characterized by resistance to all the known rust races, such as hybrids between C. Arabica x C. canephora, either spontaneously as in the HDT or man-made as in Icatú (D'Oliveira et al. 1979).
Plants of group A have also been found in C. liberica, C. dewevrei, C. eugenioides, C. congensis, etc. (D'Oliveira and Rodrigues Jr. 1961) while the E-group, characterized by susceptibility to almost all known races, includes the traditional Typica and Bourbon cultivars (Bettencourt and Rodrigues Jr., 1988).

DURABILITY OF RESISTANCE
In the last few years, some improved commercial varieties from HDT and other interspecific tetraploid hybrids gradually losing their resistance to leaf rust in some countries, due to the appearance of new virulent races (Rodrigues et al., 2000). Some genotypes of coffee varieties, however, maintain their resistance and others, although infected in the field, present an incomplete type of resistance with others heavily infected, suggesting that they probably possess a polygenic type of resistance, like the variety Colombia (Alvarado, 2005). On the other hand, some Arabica varieties like Rume Sudan and Tafarikella with low yields and classified at CIFC as belonging to the susceptible group E, showed a very high partial resistance in the field for many years (Várzea et al., 2000).
At CIFC the level of resistance of HDT derivatives and some lines of Rume Sudan are now being reevaluated. Even though several varieties of coffee that are resistant to H. vastatrix have been used for introgression purposes (Bettencourt AJ et al.,1988) such alternatives are time-consuming and do not provide durable resistance due to the rapid co-evolution of races of the fungus that harbor new virulence genes (Várzea VMPand Marques DV, 2005). Therefore, additional methods to control leaf rust in the fields are required.

CYTOLOGICAL AND BIOCHEMICAL RESISTANCE MECHANISMS
During incompatible interactions with biotrophic pathogens, the plant resistance phenotype results from the onset of a complex and multilayered-defense response, which is the so-called hypersensitive response or HR (Cacas JL., 2010), Although little is still known about the molecular mechanisms that govern resistance to H. vastatrix, several studies have advanced the case for the existence of a HR-like phenomenon in coffee plants. Resistant varieties that were inoculated with avirulent fungal strains displayed a morphotype that exhibits many HR characteristics. These include rapid host cell death, which is localized at the infection site and that is associated with fungal hyphae collapse (Silva MC et al., 2002), callose encasement of haustoria and subsequent cell wall lignification (Silva MC et al., 2002), early oxidative burst (Ganesh D et al.,2006), and the activation of typical defense-related genes (Ramiro D et al.,2010). For a number of coffee (Coffea spp.) genotypes, the resistance is post-haustorial and is expressed by the rapid hypersensitive cell death (HR).
Cell death began to be observed around 2 days post-inoculation, in the guard cells only, or in both the guard and subsidiary cells at the infection sites in which the fungus reached the stage of appressorium or penetration hypha (Silva 1996;Silva et al., 2000Silva et al., , 2002. Death of subsidiary and mesophyll cells invaded by a haustorium was observed from the 3 rd day after inoculation. During the time course of infection, cell death spread to adjacent epidermal and mesophyll noninvaded cells, as has been generally described for other coffee resistant genotypes (Martins et al., 1985;Rijo et al., 1991).

Fungicides
There is no quick solution to this problem. The outbreaks have already occurred, and are known to proliferate into multi-year events. The usual means of keeping the fungus at lower level is no longer solutions to this problem. Since coffee rust spread over the producing regions of the world, fungicides have been used to lessen the outbreaks. Copper based fungicides are most common, but have short periods of effectiveness and applications must be timed carefully. In the end, these metallic fungicides can be detrimental to the environment and are limited in capacity to combat coffee rust. There has been some research into a more biological or organic approach to combat the rust. Certain 'hyperparasitic' fungi (that are parasitic to parasites) have been identified in nature that preys on the coffee rust fungus (Muller et al. 2009). One such fungus, known as the 'white halo' fungus, has been getting lots of press lately. Scientifically, there is some evidence that this and possibly other microorganisms can reduce the viability of the rust (Jackson et al., 2012). However, there has never been any practical application suggested or implemented for this biological control in coffee. Unfortunately, at this juncture there is no real way to implement these additional microorganisms on a large agricultural scale. Nor has their ability been proven to eradicate large rust outbreaks, at the size and severity that the industry is currently faced with.

SUMMARY AND CONCLUSION
Coffee is a vital crop in social, cultural and national economy of Ethiopia. Despite coffee plays dominant role in social, cultural and national economy, the country's coffee industry is potentially at risk due to biotic and abiotic stress related with climate change. Among biotic stress like disease; coffee leaf rust (Hemileia vastatrix) is one of the major disease which has been causing high crop lose worldwide including Ethiopia. Ethiopia as the source of origin for perhaps both, the host and pathogen, plays an important role in science either for breeders or pathologists. Knowing the biology of the pathogen and epidemiology of the disease is vital for designing mitigation from its hazardous effect. The assessment study revealed that Coffee leaf rust occurs in Ethiopia in nearly all areas and under all growing systems like wild, forest, and garden and plantation coffee. For further genetically, morphological and phytopathological investigations on the host and pathogen the last ecosystems of rainforest/wild coffee in Ethiopia urgently need to be protected.
In the last decades, considerable success has been attained in the use of classical breeding for resistance to control economically important plant diseases, such as coffee leaf rust. However, in the last few years, some improved commercial varieties are gradually losing their resistance to rust due to the new increase of H. vastatrix virulence races. This shows the importance of re-evaluating existing varieties with the new rust races and the care needed before the release of new ones. Priority must also be given to the identification of sources of more durable resistance. It has long been recognized that more effective durable forms of disease control might be devised if we had a better acknowledge of both the dynamics of the pathogen populations and the factors that determine host resistance or susceptibility.
In bringing different research approaches together (genetics, molecular biology, cytology and biochemistry), important advances have been made recently in coffee resistance, particularly against leaf rust. In the future, significant contributions can be expected to improve the durability of coffee resistance to both diseases that take into account the environmental needs and the development of a sustainable coffee economy.