An overview of physiological specialization of coffee leaf rust: New designation of pathotypes

Introduction

Coffee originates in Africa, more specifically in the regions of Café and Enária, in Ethiopia, where it occurs spontaneously (Mc Cook, 2006) [52] . Commercial cultivation took place with the Arabs, who later took the coffee crop to Egypt in the 16th century and then to Europe in the 17th century. From the European continent, the grain spread to the rest of the world, establishing production in the newly conquered territories in South, Central and Caribbean America. Since then, drink consumption of coffee has increased around the world, breaking records year after year. According to the International Coffee Organization (ICO), the world's largest coffee producing countries in decreasing order in 2020, were: Brazil, Vietnam, Colombia, Indonesia, Ethiopia, Honduras, India, Uganda, Mexico and Peru. financial help from other producing countries in Central America and an international agency. Around 1979, the Nicaraguan government spent more than $ 20 million to control rust. In 1980, the government initiated a program to renew the coffee crop in the country, but the rust spread quickly to the new plantations (Waller, 1982) [75] . In the 1970s-90s, the rust advanced to other countries in South America, Central America and Mexico (Mc Cook, 2006; Schieber & Zentmeyer, 1984, Avelino et al., 2015) [52,[60][61]4] . In Central America, leaf rust has caused one of the worst epidemics in the producing countries. According to data from Promecafé, the effect of rust on regional coffee production in the 2012/13 crop year was estimated at 2.7 million bags, costing around US $ 500 million. On average, more than 50% of the entire Central American coffee park, has been affected by the disease. The disease incidence rates were as follows: El Salvador 74%, Guatemala 70%, Costa Rica 64%, Nicaragua 37% and Honduras 25% (Avelino et al., 2015) [4] . In Honduras, the damage caused by the disease was low, due to the use of the rust-resistant cultivar Lempira in the main regions of the country. However, in 2017, the qualitative resistance of cv. Lempira was supplanted by the new races of the pathogen. In addition to economic losses, there was also a significant social impact. Most of Central America's coffee belong to small coffee growers, who did not have the financial means to absorb the expected losses. It is estimated that around 374,000 jobs disappeared in 2012/13 due to rust, as the labor used for the harvest would not be necessary. In addition, in many areas the loss of revenue by coffee growers was reflected in food security. The rust epidemic in Central America has also caused greater migratory pressure towards North America. In terms of the world coffee market, Central America (excluding Mexico) produced 15.8 million bags in 2011/12, accounting for about 12% of world production. The expected losses for 2012/13, with an estimated volume of 2.7 million bags, would represent a decrease of 17.1% in relation to the previous year. This situation, moreover, had significant consequences for consumers of specialty coffees, given the great importance of Central America, as a source of quality washed Arabicas. In Central America, this fungus caused major damage which implicated loss jobs of around 1.7 million coffee workers and loss income around $3.2 billion in 2017. Currently, leaf rust still causes severe damage to coffee growing in the countries of South America ( Brazil, Ecuador, Peru, Venezuela and Colombia), Central America (Guatemala, Honduras, Nicaragua, El Salvador, Costa Rica and Panama) and the Caribbean (Dominican Republic) and Mexico. The vertical or qualitative resistance of varieties originated from the crossing between Híbrido de Timor (HdT) and commercial varieties (Caturra, Catuaí, Vila Sarchi and Mundo Novo) grown in these countries, has been suplanted. Growers that have planted resistant or suscetible varieties selected in their countries are spraying against leaf rust with protector (coopper compounds) and systemic fungicides (triazols and or strobilurin). Therefore, chemical control of the disease has been used by coffee farmers in these countries (Zambolim, 2016; Zambolim & Caixeta 2018) [78,79] .

Distribution of coffee leaf rust in the world

Coffee rust probably originates in southwestern Ethiopia and was identified in 1860. The pathogen is a biotrophic fungus that attacks only plants of the genus Coffea. In the 1860s, the fungus was accidentally transported from East Africa to Ceylon (Sri Lanka). The dense monocultures of Ceylon coffee with favorable temperature and humidity, made it a place especially conducive to the epidemic. In the following decades, the epidemic spreaded across the Indian and Pacific Oceans. The disease hit coffee culture in the Philippines, Madagascar and Java (Mc Cook, 2006) [52] . In the early 1900s, Dutch producers introduced Coffea canephora var. robusta, in the Dutch East Indies, known as robusta coffee, considered to be resistant to rust. Robusta therefore became an important crop in Java, Uganda, Madagascar, Portuguese and French West Africa (Cramer, 1957; Clarence-Smith, 1870-1914) [21,22] . After 1905, the Old World began to recover its share of world coffee production -but most of it was robusta coffee. Around 1950, coffee growers in Africa, Asia and the Pacific -where rust was present -produced low quality and low value "robusta". The coffee-producing countries of the Americas up to that time, still free of rust, produced high-quality Arabica coffee. The epidemic had been effectively postponed since 1930s. In the 1950s and 1960s, rust advanced across West Africa. The region's two largest coffee producers -Côte d'Ivoire and Angola -were affected in 1954 and 1966, respectively. Since most of the coffee grown in the region was C. canephora, the rust did not affect the total coffee production. However, the epidemic has become very close to the Americas. Therefore, According to Mc Cook (2006) [52] , coffee rust spread in the Asia and Africa in the following countries: Ethiopia in 1860; Sri Lanka (Ceylon) in 1860; Philippines, Madagascar, Java and India from 1860-66); Ivory Coast in 1954 and Angola in 1966. In the 1950s and 1960s, as the epidemic spreaded across West Africa, some scientists began to express concern about the risk of coffee in the Americas. Hence, phytopathologist Frederick Wellman was assigned to work at the Inter-American Institute of Agricultural Sciences in Costa Rica to travel to Africa and Asia to study the rust epidemic on these two continents. In his report, he concluded that the western hemisphere had escaped the disease "more by luck than by any other factor". In addition, he recommended that researchers in the Americas begin to grow or select coffee plants resistant to rust or explore other forms of disease control (Wellman, 1957) [22] . In response to this recommendation, in 1955 the government of Portugal created the Coffee Rust Research Center (CIFC), in Oeiras, financed by the United States government. The CIFC started to work on the identification of H. vastatrix races and on the genetic improvement of coffee, aiming at the incorporation of resistance in susceptible varieties. Besides, they initiated selections and hybridization of Hibrido de Timor (HdT) coffee with susceptible varieties of high commercial value such as Caturra and Vila Sarchi. HdT coffee is a spontaneous hybrid of C. arabica and C. canephora, discovered in the Portuguese colony of East Timor, in the 1920s. In the late 1950s, HdT plants was taken to Oeiras, Portugal by CIFC researchers. From the CIFC, improved descendants of HdT were sent to coffee breeding programs in Brazil, Colombia and Costa Rica, where they were crossed and backcrossed with local Arabica coffee (Avelino et al., 1999) [5] . In Latin America, productive Arabica varieties were grown such as Caturra, Bourbon and Catuaí, but they are very susceptible to leaf rust. With the establishment of rust in the new areas of West Africa, Wellman reported that there was 'an increasing danger that rust would spread throughout the Americas'. For the first time, areas attacked by rust are in the direction of the winds and storms that link rust-contaminated African countries to coffee plantations in the Americas (Wellman, 1957) [22] .

As warned, rust spread from West Africa to Brazil. In January 1970, Brazilian researcher Arnaldo Gomes Medeiros identified coffee rust in Bahia state. Four months later, the disease was spreaded to all coffee growing states in Brazil, scattered by the wind (Chaves et al., 1970) [19] . So far, it is not clear how rust reached the New World. Even though wind currents spread rust over long distances in Africa, some scientists have doubted its ability to travel across the ocean. Once the disease settled in Brazil, it spreaded very quickly. The disease had taken 50 years to cross Africa from east to west; but it spreaded across the Americas in less than 15 years. As in previous outbreaks, wind was the biggest factor in the expansion of H. vastatrix spores at the local and regional level. In the late 1970s and early 1980s, coffee leaf rust spread beyond its focus in Brazil. From the Brazilian focus, it advanced over some areas of Paraguay and Argentina, in 1972, and then, significantly, to plantations in Acre, in the extreme west of the Brazilian Amazon, in 1975. The rust may have been carried from Acre to Bolivia in diseased seedlings. From Bolivia, the disease apparently went to the north of the Andes, possibly carried by seedlings or by the wind. The first country in Central America where leaf rust was detected was Nicaragua, in 1978, which has been growing coffee since the late 1800s (Stephanie, 2015) [65] . In the 1970s-90s, leaf rust advanced to other countries in South America, Central America and Mexico (Mc Cook, 2006; Avelino et al., 2015) [52,4] . Leaf rust in the Americas in addition to Brazil in 1970, was reported in the following countries: Peru 1979 (Mc Cook 2006) [52] ; Guatemala 1980 (Mc Cook 2006) [52] ; Ecuador 1981 (Mc Cook 2006) [52] ; Mexico 1981 (Schieber and Zentmeyer 1984) [60][61] ; Colombia 1983 (National Coffee Research Center -Cenicafé Colombia); Cosa Rica 1989-90 [54] (Mc Cook 2006; Avelino et al. 1989) [52,5] ; Honduras 1995-96 (Mc Cook 2006) [52] ; Nicaragua 1978 (Schieber et al., 1984) [6061] and El Salvador 1989 (Mc Cook 2006) [52] .

Identification of Hemileia vastatrix races

For race characterization, samples of uredospores of H. vastatrix were collected from diseased leaves of Coffea spp. and interspecific hybrids in the coffee grown field, coffee species collections and greenhouses. Each sample of uredospores of H. vastatrix can be stored for a couple of weeks in ampoules maintained inside a desiccator contained a sulfuric acid solution (density of 1.8 and concentration of 32.6%) to maintain the relative humidity around 50% at 5 0 C. The uredospores can also be kept in glass ampoules in liquid nitrogen at -196 ° C (Zambolim and Chaves 1974) [76] or at -80 0 C inside glass ampoules in a frezer. Next step is to get monopustule of the fungus for each sample collected. Coffea arabica var. Caturra leaves with 3 months old should be inoculated with uredospores of H. vastatrix either diretly from the field or with uredospores stored at 5 0 C, liquid nitrogen or freezer at -80 0 C. The incubation is generally at 22 0 C with relative humidity approximately 100%. After 48 hours, the inoculated plants is taken to a chamber at 22 0 C with 16 h of fluorescente light and 8 h in a dark. The isolated pustules on the leaves is harvested separately to be inoculate again in Caturra variety in order to obtain the amount of uredospores necessary for physiological race characterization. Only uredospores with a viability superior to 30% are considered appropriate for the inoculation tests. The inoculations can be done directly on the leaves of the differential clones. The inoculation of the uredospores from each multiplied monopustule of H. vastatrix is individually inoculated with a dried spores using camelhair brush or a suspension of 10 5 spores/mL with manual sprayer, over the abaxial surface of the leaves of each coffee differential clone proposed by CIFC (Table 1 and2). Distinct races or pathotypes of H. vastatrix have been identified through the differentiation of isolates of the pathogen on a set of coffee host materials with different resistance gene combinations (D' Oliveira, 1954-57) [25] . Physiologic races of plant pathogens are identified by their interaction with a set of host genotypes termed differentials (Flor, 1971) [36] . Currently, 23 coffee clones compose the differential coffee set (Zambolim & Caixeta, 2018) [79] . These clones was established by CIFC at Oeiras, Portugal (Rodrigues et al. 1965; Noronha-Wagner and Bettencourt, 1967) [53] and sent to several research institutions in different continents in the world such as Asia, Africa, South America (Brazil) and Central America (Costa Rica). The clones are mantained by vegetative propagation from the original collection of coffee leaf rust disease centre at CIFC. The characterization of the physiological races of H. vastatrix is based on the readings of the phenotypic expression (absence or presence of uredospores) in the pustule on the inoculated coffee leaves or leaf discs (Eskes 1982) [30] . The evaluation starts when the first uredospores formed on the susceptible control (Caturra). Four evaluation should be done at 7-day intervals and the recorded results is compared with the races and differential clones designated by CIFC (Rodrigues et al., 1965 [53,58,72] .

Genetic diversity of Hemileia vastatrix

The geographical distribution of H. vastatrix races seems to be dependent on the coffee genotypes planted locally, and the prevalence of certain races in a given area thus seems to occur accordingly (Bettencourt, 1981) [8] . Races of H. vastatrix identified in the Asia and Africa are on the Table 3. Twenty four races (I, II, III, VII, XI, XII, XV, XVI, XVII, XX, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXX, XXXIII, XXXIV, XXXIX, XLI, XLII) was reported in Asia and Africa since the decade of 1930 (Table 2). However, the number of races could be much greater then the 24 reported. A survey did by D'Oliveira & Rodrigues (1961) [27] and Rodrigues et al. (1965) around the world related 45 and 54 races, respectively, but they did not mention the races. In India, the apparent loss of resistance in coffee cultivars highlighted the attention of Mayne in the early 1930s. By means of experimental inoculations, he was able to differentiate, with local rust samples and host differentials, four physiologic races of H. vastatrix (Mayne 1932; 1942; Meyer 1965) [48][49] . India was the country that registered the highest number of H. vastatrix races in the world, being more than 37 (Prakash et al., 2005; Prakash et al., 2015) [54][55] . However, the number of races today could be more than 50. This country holds the most ancient breeding programme for coffee leaf rust resistance in the world, involving regular and massive introductions of new or experimental resistant coffee materials into the field, to which usually follows the appearance of new pathotypes with enlarged virulence spectra (Várzea and Marques, 2005; Prakash, 2005) [72,55] . The most complex races has been identifyied in India due to the cultivation of germplasm of coffee varieties derived from HdT (Prakash, 2005; Prakash et al., 2015) [54][55] . In 1952 a world survey of coffee leaf rust was initiated by Branquinho D' Oliveira from Coffee Rusts Research Center (CIFC), in Oeiras, Portugal (D' Oliveira 1954-1957) [25] . The work enabled the characterization of about 45 [27,28,58,59,7,71,72] . Rodrigues & Bettencourt (1975) [58] related 32 races and Gichuru et al. (2012) [38] reported 49 races. Considering that Ethiopia is the center of origin of C arabica and most probably of H. vastatrix as well, it is probable that host and parasite have undergone a parallel evolution, and that the work of natural selection has given rise to resistant plants on one side and to a number of physiologic races of the pathogen on the other (D' Oliveira, 1951) [24] . However, the screening of 66 rust samples received from several areas of Ethiopia on different occasions (D' Oliveira 1951; Bettencourt; Lopes, 1966) [24,10] led only to the identification of races I, II, III, and XV. In a study on the decade of 1950-60, Rodrigues et al. (1965) and Bettencourt & Lopes (1966) [10] identified 24 physiological races, using 13 differentiating coffee clones. Four of them, races XII, XIV, XVI and XXIV were obtained from cultures of H. vastatrix already established by the CIFC, in which they existed as a mixture or were developed through mutation or heterokaryosis (Bettencourt & Carvalho, 1968) [11] . Following the survey of phsiological races of H. vastatrix around the world, carried out by the CIFC, with 779 rust samples from over 30 different regions of the world and with a vast collection of coffee germplasm, led to the differentiation of a total of 30 physiologic races (Rodrigues et al., 1965) on a set of 17 differential hosts. In the America continent (South and Central Amércia, Caribean and México), 18 races (I, II, III, X, XV, XIII, XVI, XVII, XXII, XXIII, XXIV, XXV, XXIX, XXX, XXXIII, XXXVI, XXXVII and XXXIX) were reported (Table 4). The most comum and widespread H. vastatrix race II in the America continent acquiring a generalized occurrence, probably is due to the uniform genetic background of most C. arabica cultivars planted in the field. Race II reflected the susceptible varieties cultivated in South and Central America, Caribean and Mexico. When HdT and interespecif hybrids from CIFC was cultivated in the field in Brazil and Central America, Caribean and Mexico in trials and in coffee collections, other complex races appeared (Talhinhas et al., 2016; Zambolim & Caixeta, 2018) [67,79] . Selection pressure exerted by coffee resistance genes (HdT and other interespecific hybrids derivatives) probably explain the great variability of H. vastatrix races in Brazil and other countries. The virulence profile characterization, particularly of isolates infecting HdT derivatives, can only be performed if genotypes from the collection of differentiating clones can differentiate them. The unavailability of differential coffee genotypes results in many pathotypes with incomplete characterization or entirely unidentified. As presumed from the dynamic host-pathogen co-evolutionary arms race, in which short-term selection of pathogen strains with fitness advantages is promoted, new pathotypes with increased virulence have been continuously appearing. Currently, more than 50 H. vastatrix physiological races have been identified by using coffee differentials clones of CIFC, in several countries: Portugal, India, Kenya, Indonesia, Etiopia, Tanzania, Costa Rica, Honduras, Mexico, Colombia, Venezuela, Guatemala, El Salvador, Nicarágua, Dominican Republic and Brazil (Zambolim et [54,72,38,67] ; Talhinhas et al., 2017 [68] ; Zambolim & Caixeta, 2018) [79] . Although there are many reports relating 50 races of the pathogen in the world, even though the number could be higher, there is no comprovation yet.

Genes for coffee resistance and for Hemileia vastatrix virulence H. vastatrix races are identified and characterized in a coffee host material bearing different resistance gene combinations under prescribed testing conditions (Table 1). Races are described as sequential roman numerals in order of detection (D' Oliveira, 1954-57 [25] ; Noronha-Wagner and Bettencourt, 1967) [53] . Noronha-Wagner and Bettencourt (1967) [53] , by allelism test and inoculation of different coffee plants and the progenies from their crosses, found four resistant genes and named them as SH. Using 12 physiologic races of H. vastatrix, they identified the gene SH1 in clone 87/1 Geisha, SH2 in 32/1 DK 1/6, SH3 in 33/1 S.288-23 and SH4 in 110/5 S4 Agaro. Some combinations of these genes have also been found in other clones. Further studies identified the genes SH5, SH6, SH7, SH8 and SH9, alone or in association, in different coffee clones (Bettencourt & Noronha-Wagner, 1971; Bettencourt et al., 1980, Bettencourt et al., 1992). Table 1 shows the coffee clones and their respective SH genes.

In the patossystem Coffea-H. vastatrix, the sexual stage of the rust is not known. Therefore, it is not possible to analyze the genetical bases of the pathogenicity. The virulence genes (v) in the pathogen are inferred based on resistance gene (SH) found in the differential coffee set, according the Flor's gene-to-gene theory (Noronha-Wagner and Bettencourt, 1967) [53] . Thus, as no further genetic confirmation has been possible so far, inferred rust race genotypes comprise virulence genes ranging from v1 to v9 in isolates derived from C. arabica and tetraploid interspecific hybrids, whereas those of the races that attack diploid coffee species are not known. Given this direct correlation with the coffee host resistance genotypes, virulence genes v1, v2, v3 and v5 can be traced back to an Arabica-type origin (v1/SH1 from 'Geisha'; v2/SH2 from 'Kent's'; v4/SH4 from 'Kaffa'; v5/SH5 from nearly all cultivars). The v3/SH3 was indentified in genotype with C. liberica introgression. The genes v6 to v9 reflect the additional C. canephora heritage of SH6 to SH9 genes present in HdT and other interspecific hybrids. HdTs are natural hybrids between C. arabica and C. canephora and received from the latter the major genes responsible for rust resistance (SH6, SH7, SH8, SH9) and others not yet identified biologicaly (Rodrigues et [58,72] . However, so far, we do not know the genes for resistance and virulence of the differentials host 849/1 Matari, C. dewevrei excelsa Longikoi (168/12,), C. congensis Uganda (263/1), C. canephora Uganda (829/1, 681/7 and 162113) and 644/18 Kawasari Hybrid (Table 2). Within this context, H. vastatrix races are atributed to isolates with distinct and unique combinations of virulence genes as inferred by Flor's gene-to-gene theory (Flor 1942, 1971) . Accordinly to Flor (1942) [32] , "for each dominant gene responsible for resistance in the host there is a dominant specific gene for avirulence in the pathogen". He also mentioned that, for Melampsora lini, the infection of the virulent type appeared to be, in general, a recessive character and resistance to the rust fungi usually has been inherited as a dominant character. In 1955, Flor confirms this finding, and proposed that the resistance genes in the host could be identified by the pathogenicity of specific races of the parasite. In addition, the genes for pathogenicity in the parasite are identified by the reaction of specific genes on the varieties of the host. Acceptance of this hypothesis enables to construct hypothetical genotypes of host and pathogen in the absence of direct genetic studies by determining the reaction of a range of host varieties to a range of pathogen races. For the patossystem Coffea-H. vastatrix, this theory was applied by CIFC researchers (d' Oliveira, 1954-57 [25] ; Rodrigues et al., 1965; Noronha-Wagner and Bettencourt, 1967) [53] . Based on the CIFC's system, race II of H. vastatrix with the recessive virulent gene v5 infect C. arabica cv. Caturra that have the dominant gene SH5, but do not infect the host with the resistant gene SH1 (128/2 Dilla & Alghe). Following the same system, race XXIX with the virulent gene v5,6,7,8,9 infect host plants 63\1 Bourbon (SH5), 1343/269 (SH6), 420/10 (SH5,6,7,9), 420/2 (SH5,8) and H419/20 (SH5,6,9). Using this strategy, the reaction of susceptibility is considered to infer genes in the host and in the pathogen. Based on the Flor theory and the susceptibility found in the differential coffee clones (Table 1 and2 The resistance of the differential clones HdT 832/1 (SH 6,7,8,9,? -v6,7,8,9,?) and HdT 832/2 (SH 6,7,8,9,? -v 6,7,8,9,?) was only supplanted in India by isolates colleted in the field; but their resistance was not supplanted in any other part of the world. In America (Colombia), Cristancho et al. (2007) [23] reported that the resistance of HdT 832/1 was suplanted in the field, but this report needs confirmation. These results suggest that this HdT have other resistant genes not yet identified. More recently, Barka et al. (2020) [1] and Almeida et al. (2020) [1] , using molecular biology technique, found two different genes in HdT 832/1 and HdT 832/2. As theses genes are not yet identified in differential clones (SH1-9), here they were named as SH10 and SH11, respectively. SH10 correspond to a resistance gene analogs (RGAs) and was compleley sequenced and characterized. This gene was detected in all the differential coffee with SH6, in HdT 832/1 and in HdT 832/2 (SH 6, 7, 8, 9, ?). In addition, a conserved sequence was report in 128/2-Dilla & Alghe, previously considered to contain only the SH1 gene, and in 644/18 H. kawisari with an uncharacterized SH-gene. The obtained results suggested that the cloned SH gene loci have a characteristic polymorphism conferring different resistance phenotypes against coffee leaf rust (Barka et al. 2020) [1] . Resistante gene SH11 was cloned based on a library of bacterial artificial chromosomes (BAC) of the the differential coffee HdT 832/2 and screened using a functional marker. A disease resistance gene analogue was cloned and characterized and showed a typical plant RLK motif (Receptor-Like Knase). The analysis of the presence/absence of this gene in a set of differential coffee suggested that the cloned candidate gene is not one of the nine SH genes reported previously (Almeida et al. 2020) [1] . The main source of genes for resistance to H. vastatrix used in the world are: SH1, 2, 4, 5 -Coffea arabica; SH 3 -Coffea liberica; SH 6, 7, 8, 9 -C. canephora var. robusta and C. canephora var. conilon, and SH 6, 7, 8, 9 -Timor Hybrid. Probably there are other genes not characterized yet. The most important resistance sources (HdT 832/1 and HdT 832/2) with the genes SH 6, 7, 8, 9 were crossed and backcrossed with the elite cultivars as Caturra, Vila Sarchi, Mundo Novo and Catuaí, resulting in new resistant cultivar. However, the resistance of some of these cultivars where supplanted and were attacked by the isolates and pathotypes of the America's continent. The first hybrids using these resistance sources was obtained by CIFC. The hybrid H361 (Villa Sarchi CIFC 971/10 x CIFC HDT 832/2) was selected and seeds were distributed to many contries. The selected material of this hybrid was the starting point for further improvement and selection (Varzea et al. 2009) [38] . Progenies derived from H361 were designated as Sarchimor. Other important hybrids developed by CIFC researchers was the denominated Catimor (CIFC 19/1 Caturra x CIFC HDT 832/1). Derivatives of Sarchimor and Catimor were develeloped in breeding programs and have been under commercial cultivation in diferent coffee growing countries. However, the cultivars have lost their complete resistance to rust. Sarchimor seems to have more durable resistance than Catimor. Very few cultivar derived from Catimor are stil resistant under field conditions in Brazil.

Pathothypes of coffee leaf rust

The name pathotype has been used by many authors to refer the variability of plant pathogens (Buck, 2013; Takaoka et al. 2014; Kan-Fa (2015) ; Aghnoum et al., 2019) [13,66,44,3] . In the present review, we are proposing this name to refer the isolates of H. vastatrix that could not be differentiated into races according to the CIFC system. Surveys of approximately 225 isolates of H. vastatrix collected in Brazil, Honduras, Venezuela and Costa Rica from 2018 -2020, on HdT derivatives that lost the resistance, could not be differentiated into H. vastatrix races. It became evident that race nomenclature based on a set of differentials of CIFC was not enough to characterize complex isolates from HdT derivatives into races. For this reason, the isolates have been named as pathotypes. Pathotype is a strain of an organism that is virulent in a wide host range. They are designated when virulent genes of the pathogen do not interact differentially with the genes for resistance of the host plant. Ten (Hv01 to Hv10) and eight (Hv01 to Hv08) Hemileia vastatrix isolates have been identified in Brazil and Honduras, respectively as pathotypes due to the fact that they do not fit in any possible combination of race on the coffee clones differentials (Table 5 and6). In Colombia and Tanzania also many isolates of H. vastatrix from the field could not be differentiated into races (Gouveia et al., 2005; Kilambo et al. 2013) [40,45] . The three identified pathotypes in Brazil named Hv01 (v1, 5, 6, 7, 8, 9?), Hv02 (v1, 5, 6, 8, 9?) and Hv08 (v1, 2, 5, 6, 7, 8, 9?) have the virulence gene v8-9 pathogenic to H420/2 (SH 8, 9) and H420/10 (SH 5, 6, 7, 9) (Table 4). These pathotypes (Hv01, Hv02 and Hv08) did not infect HdT 832/1, HdT 832/2 and 644/18 Kawisari hybrid (SH ?), respectively. For these reasons, we can infer that HdT 832/1, HdT 832/2 and Kawisari hybrid might have more resistant genes in their genome. Two identified pathotypes in Honduras Hv 03 (v 1, 2, 4, 5, 8), Hv 06 (v 1, 2, 4, 5, 7, 8) and Hv 08 (v 1, 2, 4, 5, 6, 7, 8, 9, ?) has the virulence gene v8 pathogenic to H420/2 (SH 5, 8) (Table 5). But only the pathotypes Hv 01 (v 1, 2, 4, 5, 6, 7, 9,?) and Hv 08 (v 1, 2, 4, 5, 6, 7, 8, 9,?) has the virulent gene v9. These pathotypes (Hv 01 and Hv 08) although have the virulent gene v 6,7,8,9,? do not infect 644/18 Kawisari hybrid, HdT 832/1 and HdT 832/2, respectively. The pathotype Hv 02 from Honduras with the virulent gene v 1, 2, 3, 4, 5 infected the clones H 147/1 (SH2, 3, 4, 5) and 33/1 S. 288-23 (SH3, 5). These findings are very important due to the fact that cultivars containing the gene SH3 in Honduras might be attacked by the race that has the gene v3 already present in this country. In Brazil, all the surveys, so far, did not find races or pathotypes with the gene v3 (Toma-Brachini, 2015; Zambolim & Caixeta, 2018) [70,79] . The virulent genes v 8, 9 was not identified yet on the 16 identified races in Brazil (Zambolim et al., 2005; Zambolim 2016; Zambolim & Caixeta 2018) [54,78] . The clone HdT 1343/269 (SH6) was susceptible to pathtypes Hv 01, Hv 02, Hv 03, Hv 05, Hv, 06, Hv, 07, Hv, 08 and Hv 09 from Brazil and, therefore, have the virulent gene v6. It was observed that only the pathotype Hv 10 (v 1,4,5) had the virulent gene v 4. All of these pathotypes were identified after almost 50 years, since the discovered of coffee leaf rust in Brazil, was in 1970. As soon as the resistant varieties derived from HdT 832/1 and HdT 832/2 increased cultivation by coffee growers from the year 2005, the new pathotypes have been identified. Almost all the coffee growing areas cultivated in Brazil have resistant varieties; those derived form HdT 832/1 lost the resistance very rapidly and few varieties derived from HdT 832/2 still remains resistant. Two identified pathotypes in Honduras Hv 03 (v 1, 2, 4, 5, 8), Hv 06 (v 1, 2, 4, 5, 7, 8) and Hv 08 (v 1, 2, 4, 5, 6, 7, 8, 9,?) have the virulence gene v8 pathogenic to H420/2 (SH 5, 8) (Table 4). But only the pathotypes Hv 01 (v 1, 2, 4, 5, 6, 7, 9,?) and Hv 08 (v 1, 2, 4, 5, 6, 7, 8, 9,?) have the virulent gene v9. These pathotypes (Hv 01 and Hv 08) although have the virulent gene v 6, 7, 8, 9,? did not infect HdT 832/1 and HdT 832/2, respectively. These data corroborate to hypothesis that HdT 832/1 and HdT 832/2 might have more resistant genes in their genome. The virulent gene v4 was identified in only one pathotype of Brazil (Hv 10 -v1,4,5) (Table 5), but it was presented in Honduras in all the pathotypes from Hv 1 to Hv 8 (Table 6). A survey in Costa Rica isolates of H. vastatrix from 2018 -2020 found the presence of the pathotype v 1, 5, 6, 7, 8, 9, ? In Tanzania, Kilambo et al. (2013) [45] reported that at least four isolates of H. vastatrix could not be differentiated into races. This could indicate that the differential clones of Coffea spp. of CIFC were not able to confirm the isolates into races but they are very importante to distinguish the pathotypes. The genome of the pathotypes are recognized based on the interaction of the isolates of the pathogen and the host differentials. According to the results the pathotypes are designated (Table 5 and6). The pathotypes from Brazil and Honduras according to the results of Table 5 and 6 are completely different. Other pathotypes from Venezuela, Costa Rica and Ethiopia were also diffents (data not shown). Twenty host differentials was used to distinguish ten pathotypes from Brazil and eight from Honduras.

New propose of pathotypes designation

As mentioned above, in the CIFC system, the designation of the pathotype follow the gene-to-gene theory. Accordinly of Flor works (Flor, 1942, 1946, 1947 and 1955) [32][33][34][35][36] , "the inheritance of avirulence and resistance or of virulence and susceptibility has been explained as result of specific genes in the host interaction with specific gene in the parasite". However, for the patossystem Coffea-H. vastatrix, CIFC system based the characterization of the genes on resistance of the coffee varieties and virulence of the fungus, instead resistance and avirulence. Besides, the susceptibility reaction is considering to infer the host and pathogen genes. Based on the Flor theory, resistant reaction is observed only when the plant has at least one allele of resistance (RR or Rr) and the fungus has at least one allele of avirulence (AA or Aa). Susceptibility reaction occur in three situations: 1) the plant and the fungus has no resistance gene (rr) and no avirulence gene (aa), respectively; 2) the plant has resistance gene (RR or Rr) but the fungus has no avirulence gene (aa); and, 3) the plant has no resistance gene (rr) and the fungus has avirulence gene (AA or Aa). For this reason, we are proposing the fungus and coffee gene prediction using the resistance reaction and the inheritance of avirulence and resistance. We expected that this new approach would facilitate the gene inference and also, help to join the data of inference with molecular characterization of the genes. The designation of the pathotypes followed the two systems is shown in Table 7. By the CIFC system the pathogen is recessive and virulent (v1-9) and the host plants are dominant and resistant (SH1-9) and the interaction is susceptible (S). In our proposed system, the pathogen is avirulent (Avr1-9) and the host plant is also dominant (SH1-9) and the interaction is resistant (R). Following the CIFC system, the brasilian pathotype Hv01 has the virulent genes v 1,5,6,7,8,9 and infects the coffee diferentials with the resistant genes SH 1,5,6,7,8,9? On the other hand, in the new system, the pathotype Hv01 has the avirulent genes Avr1,5,6,7,8,9? and do not infect the coffee diferentials with the resistant genes SH 1,5,6,7,8,9? For the isolate Hv01 from Brazil of H. vastatrix, CIFC system is pathogen virulent gene v 1,5,6,7,8,9? and the resistant gene on the plant host is SH 1,5,6,7,8,9? (Table 7). The result is: H. vastatrix isolate is virulent on the coffee differential SH 1,5,6,7,8,9?. On the other hand we proposed a system based on Flor theory which the pathogen with the gene Avr 2,3,4? can not infect a coffee differential containing the resistant gene SH 2,3,4? (Table 7). Our proposed system is more simple and follows Flor theory straight forward. Furthermore the number of differentials of CIFC is enough to distinguish pathotypes instead of races. [25] ; Rodrigues et al. (1965) [28] ; Noronha-Wagner and Bettencourt (1967) [7] . Pathotypes from Hv01 to Hv08 were identified in the Lempira cultivar considered resistant to H. vastatrix. 1 Virulent genes according to CIFC system; 2 Virulent genes according to the new proposed system; ; 3 Means susceptible to the pathogen (S); reaction to the others pathotypes are resistant (R).

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