Research into the pathological condition affecting Cypripedium acaule in the Manitoba Model Forest

by:  Carla Zelmer   Mar. 2002

prepared for:  Mr. Mike Waldram, General Manager, Manitoba Model Forest



Introduction

This study was undertaken because of observations by members of Native Orchid Conservation, Inc. (NOCI) that moccasin flower numbers were declining in habitats in Manitoba that used to contain large, healthy populations. Of particular concern were forested sites around Belair, Manitoba which had been observed annually for 20 years. In declining populations, many stunted plants were noted, and afflicted plants often disappeared from the site in subsequent years. The cause of the decline and death of these plants was unknown, but leaf tissue damage (necrotic lesions) appearing in early to mid-summer suggested a fungal pathogen.


Objectives

The goals of this research project were as follow:

1) to determine the cause of the leaf lesions

2) to follow the fate of marked plants at 4 sites in the Belair area as an assist to detecting diseased plants

3) to survey additional moccasin flower sites in Manitoba and Ontario to determine if these leaf symptoms were restricted to the Belair sites, or were more geographically widespread.


Methods

To meet these objectives, two approaches were used. At each of four sites in Belair Provincial Forest, 10 flowering plants of C. acaule (photo 1) were photographed, marked for relocation later in the season and examined for signs of disease. At all sites, plants included both normal size and smaller-than-average sized plants, but all plants were mature as evidenced by flowering. At this time, leaf samples from adjacent C. acaule plants were taken to examine in detail for signs of disease. As well, a list of surrounding plants was prepared as an aid to characterizing the sites.

The occurrence of the leaf necrosis in other C. acaule populations was determined by examination of plants of 9 additional locations in Manitoba for symptoms of the disease. Leaves were collected from C. acaule plants in these areas for microscopic confirmation.


Results

Marked plants

The leaves collected on June 9, 2001 at the 4 sites in Belair Prov. Forest showed no obvious signs of disease. Stunted plants generally had shorter flowering stems, and more strongly folded, narrower and shorter leaves than those of the normal sized plants.

At Site 1, 5 normal height and 5 below average height plants were marked. All plants had bloomed this year, and 2 plants each of the short and normal height plants had set seed last year as evidenced by their persistent seed capsules. All plants marked at this site were single plants each with the usual set of 2 leaves, with the exception of one plant which had two sets of leaves. Diseased plants previously had been noted at this site.

At Site 2, four of the ten plants (two of each size class) were clump-forming plants with 4 leaves each. No capsules were set by the marked plants in the previous season. This site was believed to be free of the leaf necrosis disease.

As for Site 1, Site 3 plants bore single pairs of leaves with the exception of one short plant with two sets. Only one robust plant showed evidence of seed production the previous year. Deformation of the flower of one of the marked plants was noted. Diseased plants had been observed on this site.

Of the marked plants at Site 4, another believed to contain diseased plants, only 2 plants had more than one pair of leaves. No capsules were set in the previous season.


Leaves collected at the sites

Leaves from adjacent C. acaule plants were collected from the sites as the plants were marked. Dried leaves from Site 3 showed translucent patches when held to light, and several other samples from all 4 sites had minor brownish patches that could have resulted from the pressing and drying processes. Hyphae were not visible on or within these leaves under stereoscopic microscopy.

Sub-samples from all leaves from these collections were cleared in 10% potassium hydroxide to increase their translucency, and then mounted in 75% glycerin for examination under a compound microscope. Microscopically, there were no signs of fungal hyphae in the Belair collections made on June 9, 2001.


On September 15, 2001, the plants at the four sites were relocated, photographed, their conditions recorded and leaf samples collected for microscopic examination.

At Site 1, all marked plants were located. The leaves of two of the shorter-than-normal plants and three of the normal height plants had disappeared. Blackened fibrous material at the base of the plant indicated the early death and decay of these leaves. One plant had black patches on otherwise brown, intact leaves. The remaining plants had normally-senscent leaves which remained intact and turned pale brown as they dried. Two of the marked plants set capsules from this year's blooms. Both of these plants were normal height plants with unaffected to slightly blackened leaves. Leaves were collected from two of these plants for microscopic examination.

At Site 2, markers were recovered for all plants except one. Leaves of most plants at this site senesced normally, turning brown but remaining intact as the plant went into dormancy. One plant was still green at this time. Two plants, both of normal height, had blackened, decayed leaves, which were collected for further study. Two capsules were set by marked plants at this site.

Site 3 had been disturbed, perhaps raked by mushroom hunters collecting the large numbers of lobster mushrooms embedded in the duff. Eight of the ten markers were relocated. A ninth flag missing a plant number was found but could not be matched to its original plant. Despite the apparent dryness of this site, many of the marked plants, and nearby unmarked seedlings retained green leaves. Of the eight plants, two (both large plants) had blackened and decayed leaves, two showed some evidence of leaf necrosis, and four appeared healthy despite obvious physical damage to the leaves. Capsules were not formed by any of the marked plants.

At Site 4, 6 (3 normal height, 3 short) of the 10 marked plants had blackened, died and were in a final state of decay. Only dark fibrous material remained attached to the crown of the plants (photo 2). Of the remaining plants, one had leaves removed but the leaf bases were still green and appeared healthy, two were intact but senescent (eg. photo 3), and one (a clump of 4 leaves) had a few small patches of black forming on the otherwise healthy green leaves. 2 marked plants (one tall, one normal) at this site set capsules. A third flower stalk indicated the formation of a capsule which had been removed.


Conclusions from 2001 marked plant experiment

This season, we have learned that all four marked C. acaule sites, including Site 2 at which the disease had not previously been noted, had plants that suffered early leaf death and decay.

The fungus responsible for the rapid breakdown of the leaf tissues was not present on the leaves or within the leaf tissues in early June when the plants were marked. In common to all diseased leaf samples was a fungus tentatively identified as Cercospora cypripedii. This fungus was described by John Dearness of Ontario approximately 100 years ago. However, the original description is very limited, and it is not yet clear how this species differs from the other more common pathogenic Cercospora species that infect agricultural crops.

We have also determined that the reduced stature of some of the plants in these populations do not indicate their susceptibility to this disease.

The early removal of leaf area must be particularly damaging for C. acaule since only a single to two pairs of leaves are produced each year. Loss of leaf area to fungal damage would reduce the ability of these plants to restore their energy reserves before the onset of dormancy.

The death of the leaf tissue prior to natural senescence also means that the resources invested in the large leaves can not be recaptured and stored in the below-ground organs until the next season. Perhaps the stunted plants seen in these population have previously suffered defoliation and are therefore functioning on limited reserves.

The marked plants from this study remain as an opportunity to follow the development of the leaf disease and to determine the impact of the disease on the vitality and survival of the plants in subsequent seasons.



Collections from other sites

Please refer to the map of sites for the locations of these collections. C. acaule leaf samples were pressed in the field at these sites, air-dried and then subsampled and cleared for microscopic observations.

East Braintree:
Three collection were made at East Braintree. The first, on July 5, 2001 contained leaves with necrotic lesions. The lesions had a blackened centre, surrounded by a pale (chlorotic) area and then a orange-brown area (probably phenolic defense compounds produced by the plant). The small, raised pimple-like bumps within the black center zones were consistent with the disease symptoms seen at Belair. Two addition collections from separate sites at East Braintree (July 14, 2001) showed severe tissue breakdown, with blackening of the tissue and dissolution of the leaf cell structures. Raised areas on the leaves of 1 sample were producing spores (conidia) of the invading fungus.

Lee River
Collections of leaves from Lea River plants (June 19, 2001) showed macro- and microscopic symptoms similar to those of the Belair plants. One of the collections contained intact conidia and conidiophores matching the Cercospora spores from Belair. These were relatively young colonizations of the leaves.

Vassar, MB
Leaves collected at Vassar, MB (June 15, 2001) were already blackened by similar fungal hyphae to those at Belair. One sample also had larger perithecia on the leaf surface. This appears to be formed by a different fungus.

Falcon Lake, MB
No symptoms were noted on plants collected in June , 2001.

Manigotogan
At Manigotogan, C. acaule plants were symptomless at the time of sampling (June 11, 2001).

Agassiz Forest, MB
Necrotic patches on leaves collected July 1, 2001 in Agassiz Forest, MB were formed by a similar fungus to that at Belair, as well as by a number of other fungi. These plants may have been under some enviromental stress, as the tip browning seen on the leaves was not due to the presence of fungi in these areas.

Richer, MB
No symptoms were noted on plants collected on June 26, 2001.



Conclusions from leaf samples at other sites

It is evident from these collections that the disease afflicting C. acaule is not limited to the Belair Forest. It is encouraging that the disease was not discovered at all of the locations surveyed, but timing may be important in such survey work. Early in June, there were no symptoms evident on the plants of the Belair Forest. In the fall, the leaves of disease plants were so decayed that finding a plant next to a flagged marker was extremely difficult. It is not likely that such plants would be noticed on the forest floor. The early stages of colonization (late June/ early July?) are the only obvious signs of the disease, when black necrotic lesions form on the otherwise green leaves. This also emphasizes the need to follow the fate of marked plants, since the remaining unaffected plants in a diseased population may give the site a "healthy" appearance in the fall. The disease and its symptoms will need further characterization.



Future Work

There are some project funds remaining from our initial funding. We would like to apply these funds to the continuation of this project. Specifically we want to monitor the marked plants at Belair to assess survival of the plants after infection. We are also interested in continuing to determine the extent of the disease in C. acaule populations in the province of Manitoba.




Photo 1. Moccasin flower (Cypripedium acaule) in bloom.



Photo 2. Natural senescence of the leaves in fall. Although the leaves dry and turn brown, they remain intact.



Photo 3. Leaves of diseased plants die early and decay rapidly, leaving only a blackened, fibrous material that is more fungal than leaf tissue.