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

#### Mario Coca-Morante

*Departamento de Fitotecnia y Producción Vegetal Facultad de Ciencias Agrícolas Pecuarias Forestales y Veterinarias "Dr. Martin Cárdenas" Universidad Mayor de San Simón Cochabamba Bolivia* 

#### **1. Introduction**

146 New Advances and Contributions to Forestry Research

Rappé and Giovannoni ( 2003)Rappé MS, Giovannoni SJ (2003). "The uncultured microbial

Rhoades, C.C., S.L. Brosi, A.J. Dattilo, and P. Vincelli. 2003. Effect of soil compaction and

Safiyh T., Craig G., Sébastien M., Lee Newman, Adam H., Nele W., Tanja B., Jaco V., and

Shaw, C.G. and G.A. Kile. 1991. *Armillaria* root disease. USDA Forest Service, *Agriculture*

Sinclair, W.A. and H.M. Griffiths. 1994. Ash yellows and its relationship to dieback and

Sinclair, W.A. and H.H. Lyon. 2005. *Diseases of trees and shrubs*. 2nd edition. Cornell

Tjamos, E.C., Rowe R.C., Heale J.B., and Fravel D.R. 2000. *Advances in Verticillium research and disease management*. The American Phytopathological Society, St. Paul, MN. 376p. Whitman WB, Coleman DC, Wiebe WJ (1998). "Prokaryotes: the unseen majority". *Proceedings of the National Academy of Sciences of the United States of America* 95 (12): 6578–83. Wells, J.M., B.C. Raju, H.Y. Hung, W.G. Weisburg, L. Mandelco-Paul, and D.J. Brenner. 1987.

http://www.theguardians.com/Microbiology/gm\_mbr10.htmhttp://www.ancient-tree-

http://www.microbiologyprocedure.com/microbial-products-influencing-plant-

http://www.plantmanagementnetwork.org/pub/php/review/biocontrol/

*Xylella fastidiosa* gen. nov., sp. nov.: Gram-negative, xylem-limited, fastidious plant bacteria related to *Xanthomonas* spp. *International Journal of Systematic Bacteriology* 

moisture on incidence of phytophthora root rot on American chestnut (*Castanea* 

Daniel v. (2009) Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees. *Applied and Environmental Microbiology*, Vol. 75, No. 3, p. 748-757 doi: 10.1128/AEM.02239-08

majority". *Annual Review of Microbiology* 57: 369–94.

*Handbook* 691, Washington, DC. 233 p.

University Press, Ithaca, NY. 680p.

http://www.theguardians.com/Microbiology/gm\_mbr10.htm http://www.voanews.com/english/news/a-13-2009-02-13-voa31- 68673292.htmlhttp://en.wikipedia.org/wiki/Bacteria

hunt.org.uk/ancienttrees/treeecology/fungi.htm

growth/biological-control-of-plant-diseases.html

37(2):136-143.

http://en.wikipedia.org/wiki/Virus

http://www.dpvweb.net/intro/index.php http://www.forestpathology.org/bacteria.html

*dentata*) seedlings. *Forest Ecology and Management* 184:47-54.

decline of ash. *Annual Review of Phytopathology* 32:49-60.

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 *queñua* or *quehuiña* (*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 (Fjeldså & Kessler 2004). Indeed, its continued existence is threatened (Rivera 1998; mentioned by Daza von Boeck 2005).

The SNP occupies some 100,000 ha, with its *queñua* forest forming a belt around it (Daza von Boeck 2005). The work reported in this chapter focuses on an area on the southwestern side of District 3 of the SNP's *queñua* forest (to the northeast of the Oruro Department in the Sajama Province, on the western *Altiplano*, covering part of the Curahuara de Carangas and Turpo jurisdiction) (Fig. 1). Located between 68°40S-69°10W and 17°55S-18°15W, the SNP lies at an altitude of 4200-6600 m in the mountains below the Sajama Peak (6524 m) (Fig. 2) (Daza von Boeck 2005). The mean annual temperature in the area of the *queñua* forest is 10oC; the maximum temperature reached is 22oC, but winter temperatures can be as low as -30oC. The mean annual rainfall is 280 mm, with a range of 90-400 mm. The study area in District 3 lies at an altitude of 4200-4300 m.

#### **2. Conservation status of the** *queñua* **forest**

The SNP's *queñua* forest has become fragmented over centuries of human activity, leaving its animal and plant biodiversity seriously threatened (Argollo *et al.* 2006). According to

Assessment of *Lepthosphaeria polylepidis* Decline in

*Polylepis tarapacana* Phil. Trees in District 3 of the Sajama National Park, Bolivia 149

A

Fig. 2. South face of the snow-covered Sajama Peak (6542 m), view from the Patacamaya-Tambo Quemado international road. A: Geographical location of the Sajama National

(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

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*

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

Park; B: Partial view of District 3 containing the studied *queñua* forest

B

(white segmented line).

as the likely cause.

SNP's *queñua* forest.

**4. The aims of the present study were** 

plants.

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).

Fig. 1. Land sat satellite image TM (19/07/2011) showing the peak Sajama (light blue colour).

#### **3. 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

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

Fig. 1. Land sat satellite image TM (19/07/2011) showing the peak Sajama

**3. 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

*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

(Gareca *et al*. 2010).

(light blue colour).

agent.

Fig. 2. South face of the snow-covered Sajama Peak (6542 m), view from the Patacamaya-Tambo Quemado international road. A: Geographical location of the Sajama National Park; B: Partial view of District 3 containing the studied *queñua* forest (white segmented line).

(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.

#### **4. The aims of the present study were**


Assessment of *Lepthosphaeria polylepidis* Decline in

A B

*Polylepis tarapacana* Phil. Trees in District 3 of the Sajama National Park, Bolivia 151

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

Fig. 3. Signs and symptoms of decline in *P. tarapacana* trees. A: partial wilting of branches; B: totally wilted dead tree (left) and apparently healthy tree (right); C: close-up showing partial wilting; D: discoloration of the vessels; E: internal view of an apparently healthy branch (upper) and of one with wilting symptoms (lower); F: stromatic bodies under the bark; G: stromatic bodies under the bark and inside the wood; H: bitunicate asci containing

eight ascospores of *L. polylepidis* extracted from the inner stromatic bodies.

F G H

C D E

#### **5. Methodology**

#### **5.1 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.

#### **5.2 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 σ2 < µ, a randomised pattern defined as σ2 = µ, and an aggregate or clustered pattern defined as σ2 > µ, where σ2 = the variance of the size of the subpopulations, and µ = the mean size of the subpopulations.

#### **5.3 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).

#### **6. Results**

#### **6.1 Symptoms and the causal agent of decline**


Table 1 shows the proportion of apparently healthy trees, trees with black knots and trees with clear symptoms of decline that were positive for *L. polylepidis.*

+ = positive for *L. polylepidis*

Table 1. Isolation of *L. polylepidis* from *P. tarapacana* samples with decline symptoms, black knots or no disease symptoms.

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

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 σ2 < µ, a randomised pattern defined as σ2 = µ, and an aggregate or clustered pattern defined as σ2 > µ, where σ2 = the variance of the size of the subpopulations, and µ = the mean size of the subpopulations.

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

Table 1 shows the proportion of apparently healthy trees, trees with black knots and trees

**samples** 

Table 1. Isolation of *L. polylepidis* from *P. tarapacana* samples with decline symptoms, black

Apparently healthy trees 15 0%+ 6%+ Trees with black knots 15 100%+ 100%+ Trees with symptoms of decline 10 100%+ 100%+

**Potato dextrose agar (PDA)** 

**Queñua dextrose agar (QDA)** 

**5. Methodology** 

coordinates recorded.

**6. Results** 

+ = positive for *L. polylepidis*

knots or no disease symptoms.

**5.1 Examination of a permanent plot** 

**5.2 Disease assessment and spatial distribution** 

**5.3 Identification of the disease-causing agent** 

**6.1 Symptoms and the causal agent of decline** 

characterised according to Sutton (1980) and Câmara *et al*. (2002).

with clear symptoms of decline that were positive for *L. polylepidis.*

**Sample Number of** 

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

Fig. 3. Signs and symptoms of decline in *P. tarapacana* trees. A: partial wilting of branches; B: totally wilted dead tree (left) and apparently healthy tree (right); C: close-up showing partial wilting; D: discoloration of the vessels; E: internal view of an apparently healthy branch (upper) and of one with wilting symptoms (lower); F: stromatic bodies under the bark; G: stromatic bodies under the bark and inside the wood; H: bitunicate asci containing eight ascospores of *L. polylepidis* extracted from the inner stromatic bodies.

Assessment of *Lepthosphaeria polylepidis* Decline in

**6.2 Black knots on branches and their cause** 

PDA.

A

bodies.

G

*Polylepis tarapacana* Phil. Trees in District 3 of the Sajama National Park, Bolivia 153

liberated their spores in a creamy-coloured cloud. The spores were small and hyaline (Fig. 4G). According to Sutton (1980), these are characteristic of *Phoma* spp. growing on QDA and

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

B C

Fig. 5. A: black knots (stromatic bodies) on branches of *P. tarapacana*; B and C: close-up to the stromatic bodies; D: vertical section of conidioma showing layers with brown-melanised cells of scleroplectenchyma tissue containing mature asci within; E: ascus and ascospores of *L. polylepidis*; F: ascospores of *L. polylepidis*; G: isolation of *L. polylepidis* from apparently healthy branches (left) on QDA, and from a sample with stromatic bodies on PDA; H: stromatic bodies formed on PDA after 12 days; I: ascospores extracted from these stromatic

H I

D E F

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).

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

Fig. 4. Anamorphic state of *L. polylepidis* with samples showing symptoms of decline in the moisture chamber experiment. A: pieces of a branch of *P. tarapacana* showing decline symptoms; B: vascular discoloration symptoms in branches with decline; C: stromatic bodies under the ritidoma; D: samples in the moisture chamber; E: samples in the moisture chamber after 72 h of incubation at 24ºC; F: close-up of stromatic bodies (red circle) and spore cloud leaving the pycnidial structure of the anamorphic fungus (*Phoma* spp.) (Red colour arrows); G: Spore cloud leaving an ostiole (green arrow); the blue arrow shows the pycnidial body; H: pycnidial squash showing the exiting spores: I: small spores and elliptical conidia; note the almost hyaline nature of the anamporphic fungus characteristic of *Phoma* spp.

liberated their spores in a creamy-coloured cloud. The spores were small and hyaline (Fig. 4G). According to Sutton (1980), these are characteristic of *Phoma* spp. growing on QDA and PDA.

#### **6.2 Black knots on branches and their cause**

152 New Advances and Contributions to Forestry Research

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).

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

A B

D E

Fig. 4. Anamorphic state of *L. polylepidis* with samples showing symptoms of decline in the moisture chamber experiment. A: pieces of a branch of *P. tarapacana* showing decline symptoms; B: vascular discoloration symptoms in branches with decline; C: stromatic bodies under the ritidoma; D: samples in the moisture chamber; E: samples in the moisture chamber after 72 h of incubation at 24ºC; F: close-up of stromatic bodies (red circle) and spore cloud leaving the pycnidial structure of the anamorphic fungus (*Phoma* spp.) (Red colour arrows); G: Spore cloud leaving an ostiole (green arrow); the blue arrow shows the pycnidial body; H: pycnidial squash showing the exiting spores: I: small spores and

G H I

elliptical conidia; note the almost hyaline nature of the anamporphic fungus characteristic of

*Phoma* spp.

C

F

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

Fig. 5. A: black knots (stromatic bodies) on branches of *P. tarapacana*; B and C: close-up to the stromatic bodies; D: vertical section of conidioma showing layers with brown-melanised cells of scleroplectenchyma tissue containing mature asci within; E: ascus and ascospores of *L. polylepidis*; F: ascospores of *L. polylepidis*; G: isolation of *L. polylepidis* from apparently healthy branches (left) on QDA, and from a sample with stromatic bodies on PDA; H: stromatic bodies formed on PDA after 12 days; I: ascospores extracted from these stromatic bodies.

Assessment of *Lepthosphaeria polylepidis* Decline in

*Polylepis tarapacana* Phil. Trees in District 3 of the Sajama National Park, Bolivia 155

Fig. 6. Incidence, spatial distribution and frequency distribution of diseased, apparently healthy, dead and burnt trees in the experimental plot. A: Incidence of disease; B:

quadrant.

**C** 

**B** 

Distribution of *queñua* trees in the plot (E=diseased tree; M=dead tree; S= apparently healthy tree and Q=burnt tree; B: Health status of the *queñua* trees in the plot; C: Frequency of healthy trees (S, blue), diseased trees (E, red), dead (M, green) and burned (Q, black) by plot

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).

#### **6.3 Incidence and spatial distribution**

Ninety eight *queñua* trees were recorded in the experimental plot. Fifty three (54%) showed symptoms of decline (sometimes with and sometimes without black knots on the branches), twenty-one (21%) trees were dead (due to diseases and other, non-established causes), and the remaining 23 (23%) were apparently healthy (Fig. 6A). The spatial distribution of plant disease (E) in the plot showed an aggregated or clustered pattern (σ2 > µ 4.3 > 2.1) (Fig. 6B). The diseased trees were distributed in 18 quadrants (72% of the total 25) and the apparently healthy trees (S) in seven (28% of the total 25) (Fig. 6C). Eleven quadrants (1, 7, 8, 9, 12, 13, 14, 16, 18, 19 and 21) had 1-2 diseased trees, and seven quadrants (3, 10, 11, 15, 17, 23 and 25) from 2-7 diseased trees (Fig. 6C).

**A** 

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

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*

Ninety eight *queñua* trees were recorded in the experimental plot. Fifty three (54%) showed symptoms of decline (sometimes with and sometimes without black knots on the branches), twenty-one (21%) trees were dead (due to diseases and other, non-established causes), and the remaining 23 (23%) were apparently healthy (Fig. 6A). The spatial distribution of plant disease (E) in the plot showed an aggregated or clustered pattern (σ2 > µ 4.3 > 2.1) (Fig. 6B). The diseased trees were distributed in 18 quadrants (72% of the total 25) and the apparently healthy trees (S) in seven (28% of the total 25) (Fig. 6C). Eleven quadrants (1, 7, 8, 9, 12, 13, 14, 16, 18, 19 and 21) had 1-2 diseased trees, and seven quadrants (3, 10, 11, 15, 17, 23 and 25)

(Fig. 5I).

(Fig. 4I).

**A** 

**6.3 Incidence and spatial distribution** 

from 2-7 diseased trees (Fig. 6C).

Fig. 6. Incidence, spatial distribution and frequency distribution of diseased, apparently healthy, dead and burnt trees in the experimental plot. A: Incidence of disease; B: Distribution of *queñua* trees in the plot (E=diseased tree; M=dead tree; S= apparently healthy tree and Q=burnt tree; B: Health status of the *queñua* trees in the plot; C: Frequency of healthy trees (S, blue), diseased trees (E, red), dead (M, green) and burned (Q, black) by plot quadrant.

Assessment of *Lepthosphaeria polylepidis* Decline in

changes in the climate favourable to the pathogen.

associated with the greater incidence of this disease.

**8. Conclusion** 

**9. Acknowledgment** 

**10. References** 

decline of *queñua* trees in District 3.

*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 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

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

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

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.

Leptosphaeria and Phaeosphaeria. Mycologia, 94(4), pp. 630-640.

Mountain Remote Sensing Cartography. Pp:-9-16.

Argollo J., Claudia Solís and Villalba R. 2006. History of High Mountain Forests of *Polylepis* 

Câmara M.P.S., Palm M.E., van Berkum P. & O'neill N. R. 2002. Molecular phylogeni of

*Tarapacana* at the Bolivian Central Andes. 8th International Symposium on High

to show several levels of severity. A worsening situation may be developing.

#### **7. 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 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.

#### **8. Conclusion**

156 New Advances and Contributions to Forestry Research

*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,

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

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

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

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

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

associated with polycyclic epidemics (Madden *et al*. 2007).

these agents depends largely on the extent of their cortical invasion.

**7. Discussion** 

totally different to those of District 3.

2006; Pinto Alzérreca 2007).

death of *queñua* trees.

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.

#### **9. 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.

#### **10. References**


**Section 3** 

**Amelioration of Dwindling Forest Resource** 

**Through Plantation Development** 

