Biological material

Hemileia vastatrix spores were collected in five plots in the Quillabamba coffee producing area (Cusco Region) and in seven plots in the Villa Rica coffee producing area (Pasco Region) (Table 1). These areas are geographically distant and considered to be among the highest coffee-producing regions in Peru (Figure 1).

In each plot, we gathered 25 leaves from coffee trees of the Typica and Caturra varieties that are infected with H. vastatrix and extracted spores. To obtain enough DNA concentration, samples from one or two plots were grouped to form a compound sample (Table 1).

Table 1 Geographic information of the plots where samples of pustules of 25 leaves of Hemileia vastatrix were collected in the two Peruvian coffee production areas; it includes keys of the compounded samples formed. 

Figure 1 Sites where leaves of coffee infected with yellow rust were collected in Peru. 

Analysis of genetic diversity in Quillabamba and Villa Rica

PCR and Purification

The ITS region was amplified using PCR and ITS1L and ITS4R universal primers (^{White et al., 1990}); as a reference, we used the protocol described by ^{Cristancho et al. (2007)}. The amplification reaction was performed in a 10 μL volume containing 2 mM of MgCl2, 0.2 mM of dNTPs, 0.4 μM of each primer, PCR 1X buffer, 1.5 units of Taq DNA polymerase and 40 ng of DNA. The amplification procedure consisted of an initial stage at 95 °C for 5 min, followed by 30 denaturation cycles at 94 °C for 1.5 min, alignment at 60 °C for 1 min and extension at 72ºC for 2 min, plus a final extension at 72ºC for 5 min. The amplification products were separated by electrophoresis in 1% agarose gels; a QIAquick gel extraction Kit® was used to purify the DNA fragment of the ITS region.

Cloning and sequencing

The purified amplified products were cloned in chemocompetent Escherichia coli (JM109) using the Pgem T-easy vector system II Kit®, following the protocol recommended by the manufacturer. Then, for each compound sample, 10 white colonies were selected and each was sown in 5 ml of LB medium with 60 μg/mL of ampicillin, and then incubated at 37 °C for 24 h. The E. coli plasmid containing the DNA fragment of interest was extracted using the protocol of the Wizard®Plus SV Minipreps DNA purification system from PROMEGA. The presence of the DNA sequence of the ITS region was confirmed through digestion with the EcoRI enzyme. The fragments were visualized in 1.5% agarose gel. The sequencing of the cloned fragments was performed by the GenBioteck company (Argentina).

Data analysis

The obtained sequences were aligned and edited using the Codon Code Aligner v 2.0.6 program (Codon Code Corporation). As a reference, we used the DNA sequences of the ITS region of H. vastatrix stored in the GenBank (^{Cristancho et al., 2007}).

The obtained sequences were aligned using the MAFFT program implemented in the UGENE platform (^{Golosova et al., 2014}). Nucleotide and haplotype diversities (^{Nei and Li, 1979}) were calculated using the DNAsp program version 5 (^{Librado and Rozas, 2009}), and to detect similarities among haplotypes a network was created using the Median Joining algorithm (MJ) implemented in the PopArt program (^{Bandelt et al., 1999}) from which gap sites were excluded.

We also analyzed sequences of the ITS regions of Hemileia vastatrix stored in the GenBank, which correspond to H. vastatrix from Colombia, and of races II and XXII from CIFC (^{Cristancho et al., 2007}).

Analysis of the genetic diversity in Quillabamba and Villa Rica

From samples of H. vastatrix collected in plots in Quillababa (5 plots) and Villa Rica (7 plots), we obtained 106 DNA sequences of the ITS region of Hemileia vastatrix. The length of the sequences ranged between 910 pb and 918 pb.

By aligning these sequences, we obtained 921 aligned sites, of which 21 were gaps (insertions or deletions). We also found 101 polymorphic sites with 11 informative parsimonious positions (Table 2).

Table 2 Values of trameters obtained from descriptive measures of Hemileia vastatrix variability using sequences of the ITS region associated with samples from Quillabamba, Villa Rica, Colombia and CIFC. 

CIFC: Centro de Invesgação das Ferrugens do Cafeeiro.

In the two coffee producing areas, a total of 68 haplotypes was found with haplotype (Hd = 0.953 +/- 0.014) and nucleotide (Pi = de 0.00409 +/- 0.00024) diversity; 62 haplotypes showed unique sequences and 6 haplotypes consisted of more than two sequences (Table 3). Haplotypes Hap_4, Hap_8, Hap_36 and Hap_21 were found more often and are considered ancestors (Figure 2); they are also distributed across both coffee-producing areas, where they must have arrived at different times and became adapted to each area’s environmental conditions. Haplotypes Hap_4 and Hap_36 are found more often in Villa Rica, while Hap_8 and Hap_21 are found more often in Quillabamba. The distribution of these ancestors in the two areas is explained by the coalescence genetic theory also observed by ^{Santana et al. (2007)} in populations of the fungus in Brazil.

Table 3 Cont’d… Regional distribution of the occurrence of H. vastatrix haplotypes obtained from ITS sequences. 

x Hap: Haplotype.

y HVQ: Hemileia vastatrix from Quillabamba; HVV: Hemileia vastatrix from Villa Rica; CT: Samples of yellow rust from Colombia; Race XXII and Race II: Samples corresponding to CIFC.

Figure 2 Haplotype network created using the Median Joinig method for 68 haplotypes detected for the ITS region of Hemileia vastatrix from Quillabamba and Villa Rica coffee production areas. Circles represent a unique haplotype; the size of the circles is proportional to the number of sequences of a haplotype; transverse lines represent the events of simple mutations, and black nodes lost or non-sampled haplotypes. 

From the analysis of H. vastatrix populations from Quillabamba and Villa Rica, we concluded that the highest genetic diversity is present in the Quillabamba coffee producing area, because of its higher nucleotide and haplotype diversity values (Table 2).

The absence of exclusive nodes by geographic region in the haplotype network in Figure 2 would indicate a large and undifferentiated population of H. vastatrix that may have resulted from the flow of spores between the two areas carried by people, seed and/or infected plants. These results are similar to those obtained in Brazil by ^{Cabral et al. (2016)}, who did not observe that H. vastatrix populations were structured based on their origin, host or physiological race. However, the genetic diversity was relatively lower than the diversity found in the present study. However, ^{Maia et al. (2013)} (also in Brazil) obtained results similar to those of the present study using 91 fungal isolates; they proposed that H. vastatrix acts as a large indifferentiated population with high genotypic diversity that is not structured based on its geographic origin and host. Therefore, the lack of structure of the two H. vastatrix populations by region may be explained by the fact that the fungus is spread over long distances.

Tajima’s neutrality test was used for H. vastatrix populations from Villa Rica (D = -2.50181; P < 0.001) and Quillabamba (D = -2.35287; P < 0.001), and for both areas together (D= -2.66315; P < 0.001). These values are lower than 0, which indicate that H. vastatrix populations are in process of expansion. Also, they may have originated from a few recently introduced genotypes, or may descend from race II, which, according to ^{Scheiber and Zentmyer (1984)} exists in Peru since 1979. These Tajima’s D test negative values may explain the presence of a high number of unique haplotypes in the haplotype network that are increasing the variability due to accelerated population growth. Therefore, this reflects the status of the H. vastatrix population and shows that coffee yellow rust epidemics in Peru, which started in 2013, were initially caused by a few individuals. However, it was not possible to establish the effect of the altitude above sea level on the population, because the Tajima test negative value indicates that there is no evidence of the existence of a selection process. The mechanisms that increase variability and create new H. vastatrix races are unknown. Therefore, spontaneous mutations may be the main mechanism responsible for H. vastatrix variability, especially in seasons of higher disease incidence. Another reason could be the selection pressure exerted by the environment. In this regard, ^{Varzea and Marques (2005)} believe that in Brazil the presence of resistance genes in some varieties of the host species increases the number of virulent mutants in the population. Another factor that could explain the frequent and rapid emergence of new variants is cryptosexuality (^{Carvalho et al., 2011}), which may have an important role in creating H. vastatrix genetic variability.

Comparing H. vastatrix haplotypes from Peru to haplotypes from Colombia

In the analysis of the ITS sequences of Peruvian H. vastatrix populations, we included 15 DNA sequences from the ITS region of the fungus that are stored in the GenBank; of these, 12 sequences correspond to H. vastatrix from Colombia, and 3 from CIFC. The latter are race II and XXII haplotypes (Figure 3). The analysis found a total of 73 haplotypes, 65 of which were unique (they had no replications). Also, it was confirmed that haplotype Hap_4 contains the races II and XXII. It is frequently present and distributed across all the studied areas (7 sequences from Quillabamba, 13 from Villa Rica, 1 from Colombia and 2 from CIFC). Therefore, we may assume that the other haplotypes emerged from this ancestral haplotype, which confirms the findings of ^{Zambolim et al. (2005)} in Brazil, who suggested that the other races emerged from race II, which prevails in Latin America (^{Cristancho et al., 2007}). It was also observed that haplotypes Hap_36 and Hap_21, which are present in Colombia, Villa Rica and Quillabamba, are nodes that appeared later. Haplotype Hap_8 is present only in Quillabamba and Villa Rica; after being introduced or emerging in Peru, this haplotype was able to better adapt to the environmental conditions of Peru’s coffee producing areas. Based on what we observed in the main nodes (in which the presence of H. vastatrix sequences from Colombia and Peru is repetitive), it may be that in Peru and Colombia the disease emerged in the same area four years apart (^{Scheiber and Zentmyer, 1984}; ^{Buriticá, 2010}). No nodes by origin of provenance were observed in the network, which would indicate that H. vastatrix sequences from Colombia are similar to the sequences from Peru. Studies conducted in early 1970 indicate that the wind has an important role in spreading the disease. ^{Martines et al. (1975)} found uredospores with good germination viability at an altitude of more than 1000 m; it has also been suggested that the introduction and rapid spread of the fungus in South America was caused by the wind (^{Bowden et al., 1971}). Other disease spreading means have also been mentioned, for example, the disease was introduced into Brazil through plants from Africa carried by people (migrant workers) and seed (^{Schieber, 1972}). Therefore, anthropogenic causes may have had a very important role in bringing the fungus to Peru. Based on the analysis of H. vastatrix populations from the studied regions, three hypotheses about the origin of the 2013 yellow rust epidemic in Peru may be proposed. The first hypothesis assumes that a new H. vastatrix race derived from Race II was introduced in Peru and quickly adapted to the environmental conditions of coffee producing areas at altitudes higher than 1000 masl. Also, because the main ancestral haplotypes were found more frequently in Villa Rica, they must have arrived earlier in this area than in Quillabamba. However, in the haplotype network there is more than one main node that is considered ancestral (Hap_4, Hap_8, Hap_21 and Hap_36). The four haplotypes must have arrived in Peru progressively and in a short time. Haplotype Hap_4, that corresponds to race II, is more frequently present in both coffee producing areas and includes a CT_2N sequence from Colombia in its background.

The second hypothesis states that the H. vastatrix population evolved from race II and that a variant emerged that is able to infect high-altitude coffee trees. However, based on what we observed in the haplotype network (Figure 3), at least four variants corresponding to the ancestral haplotypes must have developed in hight altitude. However, two of those sequences of both Colombia and Peru (Hap_4 and Hap_36) may have been introduced to Colombia from Peru, but this is very unlikely because the movement of all plant materials is under government control (^{Cristancho et al., 2007}).

The third hypothesis states that changes in weather conditions may have favored the fungus to reproduce and spread toward areas above 1000 masl. This hypothesis is supported by what is observed in the haplotype network (Figure 3), in which 4 nodes are formed that correspond to the ancestral haplotypes (Hap_4, Hap_8, Hap_21 and Hap_36) which may have existed years ago, because they are present in both coffee producing areas of Peru, and are spreading. Also, the most frequently found haplotype is Hap_4, which corresponds to race II and has been present in Peru since 1979. This hypothesis is similar to the one proposed by ^{Cristancho et al. (2012)} in Colombia as the origin of the coffee epidemic in 2008 that affected Colombian coffee producing areas above 1400 masl.

Figure 3 Haplotype network created using the Median Joinig method of 73 haplotypes detected for the ITS region of Hemileia vastatrix from Quillabamba, Villa Rica, Colombia and CIFC. Circles represent a unique haplotype; the size of the circles is proportional to the number of sequences of a haplotype; transverse lines represent the events of simple mutations, and black nodes lost or non-sampled haplotypes.