THE BIOLOGY OF MYCORRHIZA IN HELIANTHEMUM MILL

NewPhytol. (1977)78,305-312.

        THE BIOLOGY OF MYCORRHIZA IN HELIANTHEMUM                             MILL

                     BY D. J. READ, H. KIANMEHR* and A. MALIBARI
                      Department of Botany, The University of Sheffield

                                  {Received 22 September 1976)

                                             SUMMARY
The mycorrhizal status of Helianthemum chamaecistus was assessed. Vegetative and repro-
ductive tissues of mature plants were free from infection by a fungus capable of producing
mycorrhizas and were normally free from any internal fungal infection. The mature gela-
tinous sheath of the seed was infected by a number of non-mycorrhizal fungi of which the
commonest were the saprophytes, Cladosporium herbarum and Ulocladium chartarum. The
so called 'tuberous roots' are typical ecto-mycorrhizal short roots. They develop following
infection by a soil-borne sclerotium-producing fungus. Isolates of the sclerotia and roots
produced typical mycorrhizal short roots in sterile cultures of both Helianthemum and
Betula pubeseens. This mycorrhizal associate is believed to be Cenococcum graniforme. It is
concluded that cyclic infection does not occur in Helianthemum and that the mycorrhizal
status of members of this genus is comparable with that of many woody species. The possible
ecological significance of the mycorrhizal association is discussed.

                                         INTRODUCTION
Helianthemum chamaecistus Mill, is a prostrate dwarf shrub of widespread distribution in
Europe and Asia Minor. In Britain, it is an important species of well-drained chalk and lime-
stone grassland (Proctor, 1956) where it successfully withstands extremes of drought and
nutrient deficiency. The plant is normally mycorrhizal and, in view of its widespread occur-
rence and association with stressed habitats, considerable interest surrounds the nature and
role of the mycorrhizal infection.
    In her investigation of mycorrhizas associated with H. chamaecistus, Boursnell (1950)
stated that a cyclic infection process occurred. She believed that the mycorrhizal fungus
entered the seedling at germination from the gelatinous coat surrounding the seed. It was
said to form 'tuberous' mycorrhizal short roots reminiscent of those of the ericaceous
Arbutus unedo L. and to spread from the seedling root system to all vegetative tissues of the
plant, only the embryo within the seed being free from infection. She pointed out that in-
fection in the mature tissues was often difficult to observe but asserted that, in seedlings
grown without such infection, growth ceased before emergence of the plumule and no
lateral roots were formed.
    Such a cyclic infection process would be similar to that reported by Rayner (1915) in
the Ericaceae. Since Pearson and Read (1973) have shown that cyclic infection does not
occur in ericaceous plants, Boursnell's findings in Helianthemum were re-examined. The
objectives of this study were therefore to determine the pattern of mycorrhizal infection in
the genus and to establish the identity of the associated fungi. A later paper will describe
some aspects of the biological role of the infection.
   *Present address: University of Mashad, Mashad, Iran.

                                                 305

306                                  D. J.READ^T^L.

                               MATERIALS AND METHODS
Fungal infeetion of plant parts
      All stages of the life cycle of the plant were examined microscopically for fungal in-
fection.

Infeetion in seedlings
        Entire seedlings were collected in various stages of development from Coombsdale,
Derbyshire (National Grid Ref. SK 223 744). Seed was also germinated in the laboratory
in Coombsdale soil, some of which had been partially sterilised (0.8 M Rad 7-irradiation)
to remove fungi. In order to examine the role of the sheath in the infection process, it was
removed from half of the seeds sown into both the sterile and unsterile soils. Seeds with
and without sheaths were washed with twenty changes of sterile water to remove superficial
contaminants and planted in 3-in. pots. They were kept in a growth room (16 h day, 25°C
day, 20°C niglit) and seedlings were harvested at 5-day intervals. These seedlings and those
collected from the field were then cleaned and stained for microscopic examination. They
were laid in dishes and flooded several times with water to remove excess soil. Dishes were
then flooded with 10% KOH and the seedlings were left to clear for 1-30 days, the longer
period being required for older seedlings. After the desired period, the KOH was drained and
seedlings were washed twice with distilled water, once with 10% HCl and again twice with
distilled water. Whole seedlings were then stained in 0.1% Cotton Blue in lactophenol,
mounted in glycerine and examined microscopically. The presence of external and internal
fungal infection was recorded.

Infeetion of vegetative parts of mature plants
       Thirty mature plants were collected from Coombsdale. Each was divided into roots,
stems and leaves, and samples of each tissue were cleared and stained as described above.
Thicker roots and stems were treated with 3% sodium hypochlorite after KOH treatment
(Bevege, 1968).

Infeetion of reproduetive struetures
       Ten flowers were selected from each of thirty plants growing in Coombsdale. The 300
flowers were dissected into sepals, petals and stamens, and stained as above. Later in the year,
five capsules and twenty-five seeds were taken randomly from each of thirty mycorrliizal
plants at the time of seed formation. Seeds were first soaked for 20 min to remove the
gelatinous sheath and then soaked for a further 2 days after which time the seed coat could
easily be removed from the embryo. The embryos were cut into small pieces. Capsules, seed
coat and embryo segments were cleared, stained and examined microscopically. The trans-
parent gelatinous sheaths were examined immediately after removal without clearing.

Isolation of fungal assoeiates
       Since Boursnell (1950) stated that the gelatinous sheath was the source of seedling
infection, the fungal flora of the sheath was examined in some detail. A routine procedure
was developed in which seeds were soaked in sterile water to facilitate removal of their
gelatinous sheaths which were then serially washed in twenty-five changes of sterile distilled
water. Normally forty seeds were handled at any one time. Of these, twenty were left with
their sheaths intact and twenty had their sheaths removed. Entire seeds, seeds without

Mycorrhiza in Helianthemum                                       307
sheaths and the washed excised sheaths were placed on to different agar media and incu-
bated at 25° C. As fungal mycelium grew on to the media, the hyphae were removed and sub-
cultured for identification.

Isolation from roots
       Short lateral or 'tuberous' roots oiHelianthemum were excised from freshly collected
seedlings and from mature root systems. They were dipped for 30 s in 10% calcium hypo-
chlorite solution, washed thirty times in sterile distilled water and plated on to 0.5% distilled
water agar. About 90% of all such roots were contaminated by bacteria. Hyphae emerging
from the remainder were excised and sub-cultured on nutrient agar.

Inoculation experiments
       Seed of H. chamaecistus was germinated with and without the presence of the gela-
tinous sheath in partially sterilized Coombsdale soil. Fifty seedlings of each category were
allowed to grow for 7 months in the growth room in order to determine whether normal
development of the plant occurred in the absence of the sheath. Further batches of seed of
each category were inoculated as they germinated in order to determine which fungus was
responsible for mycorrhiza formation. A range of different inocula were applied to soil at
the time of seed germination. These included gelatinous sheaths, fungal isolates of gelatinous
sheaths, excised mycorrhizal roots and fungal isolates of mycorrhizal roots. These cultures
were also kept for up to seven months in the growth room before being removed from the
soil, carefully washed with distilled water and analysed for mycorrhizal infection.
    Some seeds were also placed on to a nutrient agar medium suitable for the synthesis of
mycorrhizas in pine (Pachlewski, 1968). The process of mycorrhiza formation was observed
over a period of 4 months following inoculation with a root isolate.

                                                RESULTS
Fungal infection of seedlings
      Roots of seedlings collected from the field have a number of fungal associates (Table 1)
but predominant among them from an early stage is a species with brown septate hyphae. At
the cotyledon stage, this fungus is restricted to the root surface but later it grows between
the outer cortical cells (Plate 1, No. 1.). Young seedling roots also commonly show vesicular-
arbuscular (VA) infection. This infection becomes less easy to detect in older seedlings partly
because of the increasingly dense growth of brown septate mycelium. Thielaviopsis basicola
(Berk.-Br.) Ferraris is commonly found sporulating in the rhizosphere oiHelianthemum but

      Table 1. Types of fungal infection found on roots of seedlings at different stages of
                                          development
 Figures in brackets represent the percentage of the total number of seedlings examined
 Stage of          Number of    Number with         Number with       Number with other   Number with
 seedling           seedlings   brown septate           VA              unidentified      no infection
 development       examined       mycelium           infection            infection
 Cotyledon             26           14(54)              8(31)               8(31)            4(19)
 2-4 true leaves       39           36(92)              2(5)                4(10)            3(7)
 4 true leaves          7            2(28)              4(57)               1(14)            0
 Total                 62           52(84)             14(22)              13(21)            7(11)

308                                       D.3.READETAL.
it does not penetrate the root. By the time the first true leaves have developed, black 'tu-
berous' roots are commonly present (Plate 1, No. 2). These are sheathed by the brown sep-
tate mycelium previously seen on the roots of the developing seedling. These mycorrhizal
roots showed a marked resemblance to those formed by Cenococcum graniforme (Sow)
Ferd. and Winge in many woody species of acid mor-humus soils.
   It is not normally possible to determine the exact age of seedlings collected in the field
because their development can be restricted for long periods by water or nutrient shortages.
Study of laboratory-grown seedlings of known age, however, permits a precise analysis of
the development of infection with time (Table 2). In these seedlings, a maximum of 7% of
radicles show infection after 20 days growth and only 2% are infected after 5 days. Cotyle-
dons remain uninfected. Occasionally, hypocotyls show superficial infection but no internal
mycelium was seen in any of the cleared seedlings. There is thus no evidence from micro-
scopic observation that cyclic infection of seedlings occurs.
Table 2. TJie numbers of seedling parts showing fungal infection 20 days after germination
                   of seeds planted with or without gelatinous sheaths

Figures in brackets represent numbers of infections recorded 5 days after germination. The total number
of seedling parts examined («) is shown under the appropriate heading
                         Unsterilised soil                             Partially sterilised soil
             Cotyledon Hypocotyl Radicle       Seed coat   Cotyledon    Hypocotyl Radicle Seed coat
              n = 80 rt = 40        « = 40      « = 14      « = 96       n = 48         n = 48   « = 14
Gelatinous
Sheath           0          2         3(1)          5          0            4         1 (0)      6
Present
Gelatinous
Sheath           0          1         2(0)          0          0            1         0 (0)      0
Absent

Fungal infection of vegetative tissues of mature plants
       No internal infection could be detected in any vegetative part of the mature shoot sys-
tem of Helianthemum. Occasionally superficial hyphae were found associated with older
parts of the stem but these infections were of a kind that can be found on any ageing tissue
in the field.
    All mature root systems contained the swollen short laterals seen in the seedlings. There
were two types of such root, however (Plate 1, No. 3). The shortest laterals are normally
lustrous black in colour and have an extensive development of septate dark brown to black
hyphae. These roots appear to have their extension growth curtailed by the fungal infection.
Longer swollen laterals of relatively unrestricted growth are also present. These are much
lighter in colour and are associated with a mantle of septate hyaline hyphae. As shown in
the Plate, both types of lateral are produced singly along the main lateral roots and are
not normally found in clusters. Of the 300 X 1-2 cm length fine root segments examined,
64% had short laterals with a partially or completely developed ectomycorrhizal sheath
made up of the brown septate mycelium. Only 14% of the segments had light-coloured
mycorrhizal roots but this may be an underestimate because the hyaline mycelium is much
 more difficult to observe microscopically.

Infection of reproductive structures
       The level of fungal infection in reproductive tissues was closely related to the stage

Mycorrhiza in Helianthemum                                       309
of development of the flower. In the young open flower, superficial saprophytic infection
was occasionally evident. As the floral organs senesced, the levels of infection increased
markedly. Not surprisingly, the stigma was heavily infected from an early stage. Finally
the dead stamens and stigmas were completely permeated with mycelium.
   The wall of the three valved seed capsule is not infected while immature and still green.
Infection develops as the wall matures and turns brown, and is usually present before the
capsule dehisces.
   Seed taken from capsules at various stages of development show increasing amounts of
fungal infection. While there was no visible mycelium in sheaths if taken from green capsules
(Plate 2, No. 5), many seeds taken from mature closed capsules showed fungal infection
(Plate 2, No. 6). The first signs of infection in the gelatinous sheath appear in the vicinity
of the point of attachment of the seed to the placenta which strongly suggests that the
origin of the infection is the capsule wall.

Isolation of fungal associates
       A number of fungi were isolated from vegetative tissues and from the dying fioral
parts. Predominant amongst these were members of the genera Cladosporium, Ulo-
cladium, Alternaria, Fusarium and Penicillium.
    The numbers of seeds and sheaths yielding fungal isolates increased as the seed matured
(Table 3). This confirms the microscope observations and again suggests that infection is
occurring from the capsule wall. The fungus fiora of the capsule wall and gelatinous seed
sheath is comparable. The fungi isolated most frequently from these tissues were Clado-
sporium herbarum link, ex Fr. which produced dark olivaceous green cultures and an
Altemaria-XikQ fungus with dark walled septate hyphae (Plate 2, No. 6) which produced grey
to grey-black colonies on agar. This was identified as Ulocladium chartarum (Preuss) Sim-
mons by Dr M.B. EUis (Commonwealth Mycological Institute, Kew I.MI. 202021).
    These fungi were tirst recovered from the sheath at the point of attachment to the
 placenta. This suggests that the fungus seen during the microscopic observations was prob-
 ably Cladosporium or Ulocladium. By the time the capsule wall opens, many sheaths have
 a more general infection. This appears to arise independently at a number of positions on

Table 3. The numbers of fungal infections recorded from gelatinous sheaths, seeds without
sheaths and entire seeds placed on to malt or potato dextrose (P.D.A.) agars at different
                                  stages of development

Bacterial infections followed a similar pattern of development and, in the more mature sheaths and seeds,
bacterial and fungal infection normally occurred together.
Stage of capsule        Medium          Number of excised         Seeds without       Seeds with intact
development                             gelatinous sheath             sheath               sheath
                                         with infections            « = 120              n= 120
                                             n = 120
Immature               Malt Agar                12                       0                    13
green                   P.D.A.                  23                      10                     9
Turning                Malt Agar                37                      16                    32
brown                   P.D.A.                  51                      10                    41
Mature Brown           Malt Agar                88                      26                    11
Closed                  P.D.A.                  64                      28                    89
Mature Open            Malt Agar               119                      67                   111
                        P.D.A.                 116                      55                   100

310                                   D.3.READETAL.
the sheath, probably at points where the developing seed is in contact with the capsule wall.
   A dark-walled septate mycelium was also obtained from about 3% of the black mycor-
rhizal short roots. Apart from some superficial resemblance in appearance of the cultures,
this isolate was clearly distinct from the fungi obtained from the seed sheath. Reproductive
bodies were not formed in culture.
   No isolates were obtained from the white mycorrhizal roots.

Inoculation of fungal associates
       Young plants inoculated with gelatinous sheaths or isolates of gelatinous sheaths
did not develop mycorrhizal infection (Table 4). The fungi from the seed sheath did not
even extensively colonise the root surface with

Mycorrhiza in Helianthemum                                311
aseptically cultivated seedlings of Betulapubescens Ehrh., a typical Cenococcum host of mor-
humus soils, were inoculated with sclerotia from the rendzina habitat. Typical Cenococcum
mycorrhizae of the type described hy Mikola (1948) were produced on the birch. It is there-
fore probable that the major mycorrhizal associate of Helianthemum chamaecistus is Ceno-
coccum graniforme.
   Portions of root systems of//, canum (L.) Baumg. collected from Cronkley Fell, Durham
and of H. appeninum (L.) Mill, collected from Brean Down, Somerset also revealed Ceno-
coccum mycorrhizas.

                                       DISCUSSION
The absence of internal infection by any fungus capable of forming mycorrhizas strongly
suggests that cyclic infection does not occur in Helianthemum chamaecistus. This view is
strengthened by the failure of seedlings to form mycorrhizas if they are grown in sterile
media. Apart from the absence of mycorrhizas, these seedlings develop normally. There is
thus no evidence to support the view of Boursnell (1950) that cyclic infection is a typical
phenomenon and that it is essential for normal seedhng development. H. canum and H.
appeninum appear to have a mycorrhizal status similar to that of H. chamaecistus which
makes the possibility that cycUc infection occurs in members of the Cistaceae not so far
studied seem remote. Since it has also been shown that cychc infection does not occur in
the Ericaceae (Pearson and Read, 1973), we conclude that there is no satisfactory evidence
for the formation of mycorrhiza as a result of cyclic infection in any plant family.
     Most of the plants with which Helianthemum is associated in the grassland sward are
herbaceous and all are heavily infected with VA mycorrhiza (Read, Koucheki and Hodgson,
 1976). It is therefore not surprising that the earliest infection recorded in Helianthemum
seedlings is of the VA type. Sheathing mycorrhizas become estabHshed as the seedlings
develop and both the ecto- and endo- types of mycorrhiza can be seen in the same root
system of some specimens. Cenococcum becomes the dominant mycorrhizal fungus in the
mature plant. The pale coloured ecto-mycorrhizas which also develop are probably produced
by other fungi, though the study of Park (1970) indicates that they could be young Ceno-
coccum infections.
     The occurrence of Cenococcum as the major mycorrhizal associate of Helianthemum is of
 interest from both ecological and mycological standpoints. Worley and Hacskaylo (1959)
 have shown that black mycorrhizae of the Cenococcum type become more prevalent on pine
 in dry conditions. This may be due to the capacity of Cenococcum to grow at greatly re-
 duced water potentials (Mexal and Reid, 1973) but we do not know whether such changes
 of mycorrhizal status are beneficial to the host plant. Since Helianthemum is probably the
 most successful of the plants colonizing drought-stressed calcareous soils in Britain, it is
 possible that the success is associated with its peculiar mycorrhizal association. Species of
  the genus Helianthemum are widespread and important members of the vegetation of arid
  and semi-arid zones in the Middle East and the Sahara. Studies of the mycorrhizal status of
  plants from these habitats is in progress.
     Cenococcum forms ecto- or ect-endo mycorrhizas with an extremely wide range of host
  species of the temperate zone (Trappe, 1963) though Ferdinandsen and Winge (1925)
  believed the fungus to be restricted to mor-humus soils as did Mikola (1948), who suggested
  that the optimum pH for the fungus was 4.0. Its occurrence in rendzina soils with a pH up
  to 8.0 is therefore of considerable interest.

312                                     D.J.
    Preliminary observations suggest that those shrubs (like Thymus dnicei Ronn.) and herbs
which grow with Helianthemum in the less drought-prone areas but which are excluded on
the driest scree slopes are not infected with Cenococcum. This further suggests that the re-
lationship between Helianthemum and Cenococcum may be of considerable ecological signi-
ficance. The relationship between mycorrhizal infection and drought resistance in Helianthe-
mum is at present being investigated.

                                            REFERENCES
BEVEGE, D.I. (1968). A rapid technique for clearing tannins and staining intact roots for detection of
   mycorrhizas caused by Endogone spp and some records of infection in Australian plants Trans
   Br. mycol. Soc, 51,808.
BOURSNELL, J.G. (1950). The symbiotic seed borne fungus in the Cistaceae. Ann. Bot., 14, 217.
FERDINANDSEN, C. & WINGE, D. (1925). Cenococcum Fr., A monographic study. Den Kong Vet
   o LandAarskift 1925, 332.
MEXAL, J. & REID, C.P.P. (1973). The growth of selected mycorrhizal fungi in response to induced
   water stress. Can. J. Bot., 51, 1579.
MIKOLA, P. (1948). On the physiology & ecology oi Cenococcum graniforme Commun. Inst. For. Finl.
   36,1.
PACHLEWSKI, J. (1968). Studies on mycorrhizal synthesis of pine (Pinus sylvestris L.) in pure cultures
   on agar. Inst. Bad. Les. 345, 3.
PARK, J.Y. (1970). A change in colour of ageing mycorrhizal roots of Tilia americana formed by Ceno-
   coccum graniforme. Can. J. Bot., 48, 1339.
PEARSON, V. & READ, D.J. (1973). The biology of mycorrhiza in the Ericaceae. I The isolation of the
   endophyte and synthesis of mycorrhizas in aspectic culture. New Phytoi, 72, 371.
PROCTOR, M.C.F. (1956). Biological flora of the British Isles. Helianthemum chamaecistus Mill. J. Ecol
   44,683.
RAYNER, M.C. (1915). Obligate symbiosis in Calluna vulgaris. Ann. Bot., 29, 97.
READ, D.J., KOUCHEKI, H.K., & HODGSON, J.M. (1976). Vesicular-arbuscular mycorrhiza in natural
   vegetation systems. I. The occurence of infection. New Phytoi, 11, 641.
TRAPPE, J. (1963). Mycorrhizal hosts and distribution of Cenococcum graniforme. Lloydia, 27, 100.

                                 EXPLANATION OF PLATES

                                           PLATE 1
      No. 1. An early stage in mycorrhiza formation in a field-grown seedling. Dark-walled, re-
      peatedly septate hyphae form networks over the root surface where they are largely confined
      to the anticlinal cell walls.
      No. 2. Seedling collected from Coombsdale showing well developed mycorrhizal roots.
      No. 3. Part of a mature root system with the black mycorrhizal roots of limited growth,
      which are the commonest type found in the field, and pale mycorrhizal roots of relatively
      unlimited growth.
      No. 4. Sclerotia of the mycorrhizal fungus, Cenococcum graniforme, extracted from
      Coombsdale soil.

                                           PLATE 2.
      No. 5. Fully imbibed seeds oi Helianthemum chamaecistus taken from young capsules show-
      ing the gelatinous seed sheath which, at this stage, is free from fungal infection.
      No. 6. Part of an excised seed sheath from an older seed plated on to nutrient agar showing
      the growth of septate hyphae. These eventually yield typical cultures of Cladosporium or
      Ulocladium.
      No. 7. Part of the root system of a seedling grown on water agar supplemented with thiamine
      and inoculated with a surface sterilised sclerotium of Cenococcum graniforme. The youngest
      lateral roots are uninfected but mycorrhiza formation is beginning on the older roots.
      No. 8. A more mature part of the same seedling root system showing well developed mycor-
      rhizas of Cenococcum which are very similar to those found in the field.

THE NEW PHYTOLOGIST, 78, 2                         PLATE I

D. J. READ ET AL.—MYCORRHIZA   IN   HELIANTHEMUM

THE NEW PHYTOLOGIST, 78, 2   PLATE 2

7