Assessment of Lepthosphaeria polylepidis Decline in Polylepis tarapacana Phil. Trees in District 3 of the Sajama National Park, Bolivia

The Sajama National Park (SNP) was the first protected area (1939) in Bolivia (Fig. 1). Nowadays it is a National Park and Natural Management Area (Daza von Boeck 2005). The SNP contains a forest of the native Andean tree known as quenua or quehuina (Polylepis tarapacana Phil). Forest of this type is found only in the Bolivian Andes (Argollo et al. 2006), where it suffers from human disturbance, including tree felling, man-made fires, the grazing of domestic animals (Toivonen et al. 2011) and firewood and coal extraction (Fjeldsa & Kessler 2004). Indeed, its continued existence is threatened (Rivera 1998; mentioned by Daza von Boeck 2005).


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IUCN criteria, 10 of the 13 species of Polylepis in the Bolivian Andean region are threatened or almost threatened, the latter category being that into which P. tarapacana currently falls (Gareca et al. 2010).

Biological factors affecting the survival of the SPN queñua forest
The SNP's queñua forest is also at risk from disease. During systematic studies of Polylepis in Bolivia, Kessler (pers. comm.) observed malformations of the branches -black knots similar to those formed on cherry (Prunus sp.) and plum trees (Prunus domestica). The latter author proposed that the problem might be caused by Apiosporina morbosa (Schwein). However, morphological and molecular analyses performed by Macía et al. (2005) showed Leptosphaeria polylepidis M.J. Macía, M. and. Palm & M.P. Martin sp. nov. to be the causal agent.
Leptosphaeria Ces. & Of causes different diseases in annual species. Its anamorph states are known as Camarosporium, Hendersonia, Phoma, Rhabdospora and Stagonospora (Hawksworth et al. 1995). Leptosphaeria species are known to affect different members of the family Rosaceae www.intechopen.com (Huhndorf 1992, mentioned by Macía et al. 2005). According to Stoykov (2004), the fungal families Phaeosporaceae and Leptosphaeriaceae (Pleosporales) are mainly saprotrophs (very rarely hemibiotrophs) that affect herbaceous stems, leaves and the floral parts of different plants.
Using transects in the southwestern sector of the SNP, beginning at a place known as Huito, Pinto Alzérreca (2007) found that of 377 trees some 35% had black knots on their branches. However, no direct relationship between the abundance of these knots and plant health was reported, nor was tree mortality found to be related to the presence of the fungus. However, in the same year, a technical report published by the SNP recorded a great increase in the mortality of queñua trees, particularly in District 3, naming L. polylepidis as the likely cause.

The aims of the present study were
i. to describe the decline symptoms produced in queñua trees caused by L. polylepidis ii. to estimate the incidence and distribution of L. polylepidis decline in District 3 of the SNP's queñua forest.

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www.intechopen.com New Advances and Contributions to Forestry Research 150 5. Methodology

Examination of a permanent plot
A permanent plot of dimensions 100x100 m (correcting for ground undulations) was marked out in an area with representative tree density and with a structure and slope typical of District 3 of the SNP's queñua forest (Fig. 2). The boundaries of the plot were set using a device providing geographic positioning system (GPS) readings, a tape and compass; these boundaries ran S-N and W-E. The plot was divided into 25 segments of equal size. All queñua trees within the plot were labelled at a height of 1.3 m and their coordinates recorded.

Disease assessment and spatial distribution
The health of the queñua trees in the plot was assessed by recording the number of: i) apparently healthy trees (S), ii) diseased trees (E), iii) dead trees (M) and iv) burnt trees (Q). Trees with wilted leaves and branches and with black knots on the latter were considered diseased. The spatial distribution of the trees in each health category was determined according to Madden et al. (2007), with a regular pattern defined as σ 2 < µ, a randomised pattern defined as σ 2 = µ, and an aggregate or clustered pattern defined as σ 2 > µ, where σ 2 = the variance of the size of the subpopulations, and µ = the mean size of the subpopulations.

Identification of the disease-causing agent
Sample pieces of branches (approximately 10-20 cm in length) were collected from: i) 10 trees with branches showing symptoms of decline, ii) 15 trees with black knots on the branches and, iii) 15 apparently healthy trees (Table 1). Attempts to isolate the causal agent of disease involved placing 1 cm-long branch samples in a moisture chamber at 24ºC for 72 h, and culturing other 1 cm-long branch samples on two media i) Queñua Dextrose Agar (QDA) (queñua=250 g extract of leaves and branches), and, ii) Potato Dextrose Agar (PDA), according to the method of French and Hebert (1989). Fungi were identified using semipermanent slides with lactophenol according to Macía et al. (2005). The anamorph state was characterised according to Sutton (1980) and Câmara et al. (2002). The decline caused by L. polylepidis is characterized the yellowing of the apical leaves, followed by progressive die-back of the branches from the tip downwards, by gradual defoliation of the branches and death within a few years (Fig. 3A, B, C). Slicing the bark from partially or completely dry (dead) branches revealed a dark coloration (Fig. 3D, E), with abundant stromatic bodies visible under the ritidoma (Fig. 3F). Figure 3 G, shows black, spherical bodies incrusted in and below the bark (Fig. 3G). These spherical bodies, formed by the causal agent, contain a gelatinous mass composed of asci, ascospores and pseudo-paraphysae (Fig. 3H). These asci are bitunicate, cylindrical-clavate and contain eight ascospores with three transverse septa. The spores are brown when mature (Fig. 3H).

Symptoms and the causal agent of decline
Most of the samples placed in the moisture chamber showed randomised ostiolate pycnial bodies distributed over the bark (Fig. 4F). These were partially immersed in the bark and  4G). According to Sutton (1980), these are characteristic of Phoma spp. growing on QDA and PDA.

Black knots on branches and their cause
Black knots were found on the branches of both declining and apparently healthy trees (Fig.  5A). The stromatic bodies were spherical and compact (Fig. 5B, C). In cross section a thick, dark brown pseudo-parenchymatic wall was seen, with an ascus containing ascospores at the centre (Fig. 5D, E). Once again, the asci were typically bitunicate, cylindrical-clavate and contain eight ascospores with three clear brown septa (Fig. 5E, F). Fifteen samples of branches with black knots were all positive for L. polylepidis on DQA and DPA (Table 1). After three weeks on DPA, isolates from the black knots formed stromatic bodies (Fig.  5G, H). Inside these bodies gelatinous masses, formed by asci and ascospores were seen (Fig. 5I).
Only one of 15 apparently healthy branch samples returned a positive result for L. polylepidis (Table 1) (Fig. 5G). Black stromatic bodies were seen after 15 days of incubation on QDA (Fig. 5G); these contained an ascus mass and ascospores characteristic of L. polylepidis (Fig. 4I).

Discussion
L. polylepidis is regarded as a specific pathogen of Polylepis spp., and was recorded on P. tarapacana by Macia et al. (2005). Coca- Morante (2008) has also recorded decline symptoms and black knots caused by L. polylepidis in some P. besseri trees growing in the Sach'aloma forest (Cochabamba, Bolivia). The climatic conditions at Sach'aloma (3800 m) are, however, totally different to those of District 3.
Decline among the trees in the studied plot was shown by wilting and/or black knots on the branches, though apparently healthy trees may also have black knots. Wilting begins apically, becoming evermore extended and intense, until the tree suffers complete defoliation and death. The formation of black knots on the branches is the only sign of L. polylepidis infection on living P. tarapacana (Macía et al. 2005;Pinto Alzérreca and Robledo 2006;Pinto Alzérreca 2007).
Pinto Alzérreca (2007) indicates a lack of any direct relationship between the abundance of black knots (galls in her terminology) and the health of the plant. This author also indicated tree mortality not to be related to the presence of fungi. However, the present results indicate that the black knots on the branches plus decline symptoms are associated with the death of queñua trees.
Many of the samples with symptoms of decline that were cultured in the moisture chamber showed structures of Phoma spp., the anamorphic state of L. polylepidis (Sutton 1980;Hawksworth et al. 1990;Câmara et al. 2002). The telemorphic state of L. polylepidis would appear to cause moncyclic disease, while the anamorphic Phoma spp. state appears to be associated with polycyclic epidemics (Madden et al. 2007).
Black knots and symptoms of decline are usually seen in young branches. This is probably due to the ease with which the pathogen can gain access to and develop in their tissues. It is likely that the stromatic bodies formed by the pathogen under the ritidoma are related to the discoloration of the vasculature, a gradual consequence of the xylem and phloem becoming obstructed. Infection is therefore associated with the wilting seen in affected trees. According to Guest & Brown (1997), wilting results from the physical blockage of xylem vessels caused by the pathogen and, to some extent, the host response to the presence of the pathogen. Symptoms vary from yellowing, vascular browning, tylosis formation and the gumming of the vascular system, through to the general wilting of the plant. In other tree species, the vascular tissues and surrounding cortical tissues are also colonized by wilt pathogens such as the Dutch elm pathogen (Ophiostoma ulmi), the oak wilt pathogen (Ceratocystis fagacearum) and the persimmon wilt pathogen (C. diospyri). These pathogens are not true vascular wilt fungi, however, since their presence is not restricted to the vascular tissue. The damage caused by these agents depends largely on the extent of their cortical invasion.
The spatial distribution of disease in the plot showed an aggregated pattern. According to Madden et al. (2007), in aggregated patterns the points on a surface do not have an equal probability of being occupied by an individual i.e., the trees in the present plot do not have an equal probability of being infected. The dead trees (n=21) in the present work could have been killed by the studied disease but, of course, may have died of other causes. However, the detection of a single apparently healthy tree (7%) infected with L. polylepidis is indicative that some healthy trees are probably in the initial phases of infection. Several years may pass before symptoms become visible (generally this type of disease is associated with polyetic www.intechopen.com

Assessment of Lepthosphaeria polylepidis Decline in
Polylepis tarapacana Phil. Trees in District 3 of the Sajama National Park, Bolivia 157 epidemics) (Madden et al. 2007). According to the distribution of health category frequency by quadrants, it can be seen that the disease is very common in the plot area. If 7% of apparently healthy trees are infected, the disease may be having an important impact on the decline of queñua trees in District 3.
Decline in conjunction with black knots, at different levels of severity, was seen in 54% of the trees in the plot. However, Pinto Alzérreca (2007), who used transects in the same sector, reported 35% of 377 examined trees to show black knots. The disease therefore appears to have extended since that time. However, it is difficult to determine whether the disease is truly becoming more or less important in the SNP since no more historical data on decline symptoms or black knots are available. According to Garrett et al. (2009), if a disease becomes important in an area in which it was not important in the past, this may be due to changes in the climate favourable to the pathogen.
Climate change may indeed be having some effect on the Polylepis/Lepthosphaeria (plant/pathogen) pathosystem, and in the future may modify the spatial distribution of diseased trees. The Andean nations are likely to be among the most affected by climate change (Marengo et al. 2008). According to Vuille et al. (2003), western Bolivia can expect to experience slightly drier conditions, while Nuñez et al. (2008, mentioned by Marengo et al. 2009) suggest that northwestern Argentina and the Bolivian Altiplano will experience higher temperatures during the summer months and a 40% reduction in rainfall by the year 2100, leading to increasing aridity in the region. The changes experienced to date may be associated with the greater incidence of this disease.

Conclusion
These results strongly suggest that L. polylepidis is affecting P. tarapacana in District 3 of the SNP, causing decline and black knots on branches. Disease incidence appears to be high and to show several levels of severity. A worsening situation may be developing.

Acknowledgment
The author are very grateful to Dr. Edgar Gareca León of the Universidad Mayor de San Simon, for his valuable comments and suggestions, to the authorities and forest rangers of the Sajama National Park (Servicio Nacional de Parques SERNAP) for their help in fieldwork, and to Profesor Freddy Espinoza Colque and the students of the Escuela de Ciencias Forestales (ESFOR), Facultad de Ciencias Agrícolas, Pecuarias, Forestales y Veterinarias "Dr. Martin Cárdenas" (FCAPFyV), Universidad Mayor de San Simón (UMSS), Cochabamba, Bolivia, for their help in establishing the experimental plot and the collection of data; to Ing. Javier Burgos from Centro de Investigaciones y Servicios en Teledeteccion (CISTEL) of the FCAPFyV, UMSS for provided the satellite image; to Adrian Burton for version English review. Resources, authored by 16 researchers, describes negative and positive practices in forestry. Forest is a complex habitat for man, animals, insects and micro-organisms and their activities may impact positively or negatively on the forest. This complex relationship is explained in the section Forest and Organisms

References
Interactions, consisting of contributions made by six researchers. Development of tree plantations has been man's response to forest degradation and deforestation caused by human, animals and natural disasters. Plantations of beech, spruce, Eucalyptus and other species are described in the last section, Amelioration of Dwindling Forest Resources Through Plantation Development, a section consisting of five papers authored by 20 researchers. New Advances and Contributions to Forestry Research will appeal to forest scientists, researchers and allied professionals. It will be of interest to those who care about forest and who subscribe to the adage that the last tree dies with the last man on our planet. I recommend it to you; enjoy reading it, save the forest and save life!