Ann. appl. Biol.

(1992), 120, 225-234

Printed in Great Britain 225

Mango stem end rot pathogens - Fruit infection by

endophytic colonisation of the inflorescence and pedicel
By G . I. JOHNSON**, A. J. MEAD*, A. W. COOKE and J. R. DEAN
Queensland Department of Primary Industries, Indooroopilly and Ayr*,
Queensland, Australia
(Accepted 20 December 1991)

Summary
The stem end rot pathogens of mango (Mangifera indica), (Dothiorella domini-
cana, Dothiorella mangiferae, Lasiodiplodia theobromae (Syn . Diplodia nat-
alensis Phomopsis mangiferae, Cytosphaera mangiferae, Pestalotiopsis sp. and
Dothiorella ‘long’), as well as other fungi (including Alternaria alternata), were
found to occur endophytically in the stem tissue of mango trees prior to inflor-
escence emergence. On samples from trees with a record of low stem end rot
levels, colonisation did not extend into the most recently produced flush of stem
tissue. At a site with a history of high stem end rot levels, sequential monitoring
of inflorescence tissue between flowering and harvest by plating out small (c. 8
mm3) tissue pieces revealed, that at least some of the pathogens - Dothiorella
spp., P . mangiferae, Pestalotiopsis sp. and C . mangiferae gradually colonised the
inflorescence, reaching the pedicel tissue of young fruit - 8 wk after flowering.
Subsequently, detection frequency of the pathogens in inflorescence tissue
declined, possibly because of interference from copper residues (from field
sprays) accumulating on tissue samples. The detection frequency of A . alternata
also increased as Dothiorella spp. declined, however these changes could not be
attributed to antagonistic interactions between the two fungi.
Using larger tissue pieces (1-2 mm thick transverse sections, or a square of
tissue 25 mm2 x 3 mm thick) in isolations, endophytic colonisation by Dothiorella
spp. and P. mangiferae was detected in stem, inflorescence and pedicel tissues
of mature-fruit-specimens from two different sites, one unsprayed, and the other
regularly sprayed with copper. The fungi were detected more frequently in the
samples from unsprayed trees. Fruit from the sprayed orchard subsequently
developed a high level of stem end rot caused by D. dominicana, while a lower
level of stem end rot developed in unsprayed fruit, possibly because the latter fruit
were also extensively diseased by anthracnose (Colletotrichum gloeosporioides
Penz.). Endophytic colonisation of inflorescence and pedicel tissue was found to
be a primary route of infection for fruit which .develop stem end rot during
ripening.

Key words: Postharvest disease, mango, Dothiorella, Botryosphaeria,

Phomopsis, endophyte, stem end rot

Introduction
Stem end rots of mango (Mangifers indica L.) are caused by the anamorphs of Botryos-
phaeria spp. - Dothiorella dominicana Petrak et Cif., Dothiorella mangiferae H. et P. Syd.
**Present address: Division of Horticulture, CSIRO, 306 Carmody Road, St Lucia 4067, Queensland, Australia
01992 Association of Applied Biologists
226 G . I. JOHNSON, A . J . MEAD, A. W. COOKE AND J. R . DEAN

et But., Lasiodiplodia theobromae (Pat.) Griff. et Maubl. (Syn. Diplodia natalensis (Pat.)
Nowell), as well as by Phomopsis rnangiferae Ahmad, Dothiorella ‘long’, Pestalotiopsis sp.
and Cytosphaera mangiferae Died. (Johnson, Muirhead, Mayers & Cooke, 1989; Johnson,
Cooke, Mead & Wells, 1 9 9 1 ~ )Depending
. on the growing area, either D . dominicana or
L. theobromae can be the major cause of postharvest losses (Johnson etal., 1991a; Peterson,
1984), particularly when anthracnose (Colletotrichum gloeosporioides Penz.) is well con-
trolled (Johnson et al., 1989).
Infection of fruit by L. theobromae (as D . natalensis) has been reported to occur via
colonisation of the pedicel scar of mango after harvest (Pathak & Srivastava, 1967), or by
colonisation of remnant floral tissue during flowering and fruit set of oranges (Nadel, 1944;
Minz, 1946; Hildebrand, 1947).
Pathak & Srivastava (1967) observed a greater increase of stem end rot when the pedicel
was removed entirely, rather than trimmed to 3 cm, prior to exposure to natural inoculum
in an orchard for 8 h. This method of infection is unlikely to be responsible for postharvest
losses occurring in Australian commercial orchards where the fruit are harvested with long
stems (-2 cm), destemmed in the packing shed and immediately inverted on a conveyer
that passes beneath overhead water sprays for 20 min to remove sap. The removal of the
stem and washing the fruit would eliminate most inoculum present on the surface of the
fruit or stem at harvest (Darvas, 1982; Johnson & Cooke, 1989).
Johnson, Mead, Cooke & Dean (1991b) found that colonisation of floral remnants, fruit
and pedicel tissue of mango by Dothiorella spp. occurred during flowering and fruit set, but
noted an abrupt decline in the incidence of colonised fruit seven days later, when con-
siderable fruit drop had occurred. They concluded that the majority of fruit colonised at
fruit set was aborted early in fruit development. Sequential monitoring of infection of the
fruit-pedicel tissue revealed a return of Dothiorelfa spp. 6 wk later and concomitantly higher
levels of infection of peduncle tissue. Johnson et al. (1991b) suggested that infection of the
stem end tissue of fruit could occur from colonisation by Dothiorella spp. occurring
endophytically in the peduncle.
In modern terminology, the term ‘endophyte’ is used for living organisms detected inside
healthy plant tissue (Sieber, Sieber-Canavesi & Dorworth, 1990), and is generally restricted
to fungi causing “inapparent asymptomatic infections entirely within the tissues of plants”
(Carroll, 1986). Although microscopic evidence of endophytism has been reported (Bern-
stein & Carroll, 1977), the most common method of detection of endophytes involves a
triple sterilisation of host tissue (Petrini, 1986; Sieber ef al., 1990) to eliminate superficial
colonisers, and isolation onto laboratory media. Detection by isolation has been the standard
practice, since tissue colonisation is often sparse, making histopathological detection difficult
(Carroll, 1986).
In the work reported here, colonisation of mango stem and inflorescence tissue has
been monitored by isolation on to laboratory media, from before the emergence of the
inflorescence until fruit harvest, to determine whether Dothiorella spp. and other stem end
rot pathogens occur endophytically in mango trees, and to examine the role of endophytic
infections in the development of stem end rot.

Materials and Methods

At a site near Ayr (Rita Island), north Queensland, mango stems, inflorescences and
fruit were collected at rand0.m from trees used in previous surveys (Johnson et al., 1991b).
Two trees were sampled on seven occasions during a 16 wk interval from just prior to
inflorescence emergence until the end of fruit harvesting, at the growth stages shown in Fig.
 Endophytes cause mango stem end rot 227

1. Five branches consisting of stem, inflorescence and fruit (if present) were detached from
each tree and transported intact to the laboratory. Branches were detached five growth
flushes (over 100 cm) back from the growing point in samples collected prior to flowering,
and two growth flushes back from the inflorescence in subsequent samples (as indicated in
Fig. 1).
In addition, samples were collected less frequently from sites at Mutchilba, Childers,
Indooroopilly and Mareeba during the same growth interval as indicated in the Results.
At the laboratory, segments of the stem, inflorescence, peduncle and pedicel tissue (4-
20 mm long) were cut from the five branches per tree at the points indicated in Fig. 1.
Transparencies or photocopies were taken prior to sectioning to keep a permanent record
of each sample and the isolation points. Using photo-reduction, an accurate representation
of samples could be assembled (Fig. 1).
Tissue segments were triple sterilised by immersion in 95% ethanol for 60 s, sodium
hypochlorite (2.5% available chlorine) for 3 min and then 95% ethanol for 30 s (Petrini,
1986). They were then air-dried between sterile blotting paper. Preliminary studies showed
that severe sterilisation using the above method (triple sterilisation) reduced the number
of fungal isolates recovered and enumeration of the pathogens was easier compared with
immersion in sodium hypochlorite alone.
Tissue pieces (c. 8 mm3) consisting of the cuticle, epidermis and vascular tissue were
excised from each segment at four points and transferred to potato-dextrose agar amended
with streptomycin sulphate (40 pdml) (PDA) and incubated at 20-25°C in near-UV on a
12 h illumination cycle to encourage sporulation of the out-growing fungi. When necessary,
fungi were subcultured to Sach’s wheat-straw agar (Hebert, 1971) for identification.
However, most fungi were identified within 14 days of sampling using spore or colony
characters from PDA cultures. Selected fungi were tested for pathogenicity on detached
mango fruit previously dipped in hot water (55°C for 5 min) to control quiescent infections
of postharvest pathogens.

Fig. 1. Schematic diagram (not to scale) showing the growth stages and the isolation points on the
samples from tree 2 at Ayr. Segments were cut from the points indicated, on branch growth flushes I
to V (V = oldest growth sampled) from the pre-flowering sample, and flushes I and 11, the inflorescence,
and (later) the fruit, from subsequent samples. Isolations on to PDA from segments yielded either (D)-
no stem end rot pathogen, or (+) Dothiorella spp., detected on at least one segment (20%) from the
five branches per tree of each growth stage sampled. Progressive colonisation of the inflorescence and
fruit tissue by Dothiorella spp. occurred up until fruit fill 11, after which detection declined.
228 G. I. JOHNSON, A. J . MEAD, A. W. COOKE AND J . R. DEAN

Similar isolation techniques were used for samples from other sites, with the exception
of the late mature fruit sample from Childers and the mature fruit samples from Indoor-
oopilly, where following triple sterilisation, four X 1-2 mm thick transverse sections were
cut from the middle of younger tissue segments and plated out. This procedure was followed
to increase the probability of detecting an endophyte traversing the stem section at that
point. In more woody segments, instead of cutting a cross-section, squares of tissue c. 25
mm2 x 3 mm thick) were plated out, to enhance the chance of isolating sparsely distributed
endophytes. Isolation plates were examined after 7-14 days, and the incidence of Dothiorella
spp., P. rnangiferae and A . alternata assessed as a positive detection if the fungus was
isolated from one to four of the tissue pieces plated out from each segment. Results were
converted to the percentage incidence of detection of the fungi at each assay point by
averaging results from the five branches tested per tree. Mean total detections for a particular
sampling time were calculated by averaging results from individual assay points over the
total number of assay points for that sampling time.
In order to assess whether other endophytes such as Alternaria alternata (Fr.) Keissler
could have interfered with the out-growth of Dothiorella spp. from tissue samples, mycelium
from 7 day PDA cultures of A . alternata was macerated in 18 ml sterile water with mycelium
of either D . dominicana, D . mangiferae or D . ‘long’. Twenty-five pl of the inoculum was
pipetted into a 7 mm diameter well cut into PDA plates using a sterile cork borer, and
subsequent colony growth was monitored in a temperature X time growth study in darkness
at a range of temperatures, and under near-UV at 20-25°C (Table 6).

Results
All of the fungi known to cause stem end rot of mango, as well as several other fungi,
were recorded as endophytes from stem tissue of mango trees prior to inflorescence
emergence (Table 1). Prior to flowering, endophytes were virtually absent from the tissue
of the most recent growth flush (I) of the tree from the Mutchilba site with a history of low
stem end rot levels. Endophytes, including Dothiorella spp. and P. mangiferae, were
prevalent in the most recent growth flushes of branches sampled at the same time from
Mareeba and Ayr sites with a history of higher stem end rot levels (Johnson et al., 1989)
(Table 2 and Table 3).
At the Ayr site, endophytic colonisation of the inflorescence tissue by Dothiort.‘a spp.
extended further along the inflorescence with each successive sampling of tree 2, from after
inflorescence emergence until early fruit fill (I), when colonisation of pedicel tissue was
detected (Fig. 1). Colonisation by P. mangiferae, Pestalotiopsis sp. and C . mangiferae

Table 1. Endophytic fungi detected in mango stem tissue prior to inflorescence emergence
Dothiorella dominicana* Guignardia sp.
Dothiorella mangiferae* Alternaria alternaia*
Dothiorella ‘long’* Cladosporium cladosporioides (Fresen.)
Dothiorella aromatics; de Vres
Lasiodiplodia theobromae* Epicoccum purpurascens Ehrenb. ex
Phomopsis rnangiferae* Schlect
Cytosphaera mangiferae* Nigrospora sp.
Pestalotiopsis sp. * Colletotrichum gloeosporioides*
* Isolates shown to be pathogenic on mango. Those in the left hand column
are regarded as causes of stem end rot (Johnson et al., 1991a).
 Endophytes cause mango stem end rot 229

Table 2. Incidence of endophytic (a) Dothiorella spp. and (b) Phomopsis mangiferae in
various segments of mango stem tissue prior to inflorescence emergence. Results are the
average of five samples
Incidence (%) at
Mutchilba Mareeba
Stem end rot losses' a b a b
Stem growth flush** low high
V 20 20 0 40
IV 40 0 20 20
111 20 0 20 40
11 40 40 60 20
I 0 0 60 20
Tip 0 0 60 20
Mean total detections*** 20.0 10.0 36.6 26.6
* Stem end rot losses in three years of survey (Johnson et al., 1991a).
* * Stem growth flushes numbered I to V from youngest to oldest growth (see
Fig. 1).
*** Mean number of isolation points (averaged over six segments from five
branches) yielding the pathogen.

Table 3. Average sequential incidence of endophytic Dothiorella spp. in various segments

of the mango stem, inflorescence and pedicel tissue of five branches per growth stage from
an orchard with a history of high stem end rot losses. Growth stages listed are as illustrated
in Fig. 1
Incidence (%) of Dothiorella spp. at different growth stages
Fruit fill Fruit fill Late
Site: Ayr Pre-flowering Flowering Fruitset Young fruit I I1 mature
Days after flowering 0 14 42 57 74 112
Growth flush
V 40 - - - - - -
IV 80 - - - - - -
111 20 20 - - - - -
11 base - 20 - - - - -
mid 40 0 20 - - - -
top - 40 60 20 80 - 60
I base - 80 20 60 80 - 60
mid 20 60 40 40 40 - 40
top 80 40 80 60 100 60 20
Inflorescence
base 40 20 60 40 60 40
low 20 60 60 0 20
mid 10 40 60 0 -
top 0 0 40 0 -
tip - - 20 0 -
Side inflorescence
base 0 0 40 0 20
low 0 20 40 0 0
mid - 0 40 0 -
Pedicel 0 60 0 0
Pedicel-fruit connection - - - 0
Mean total detections* 46.6 37.5 24.5 30.0 53.8 12.0 26.0
* Average incidence (%) of Dothiorella spp. over all isolation points within a sample.
- No isolation made
230 G . I. JOHNSON, A . J. MEAD, A. W. COOKE AND J. R. DEAN

followed a similar pattern. Endophytic colonisation of tree 1 was less extensive, and the
results are not presented. After this time, successful detection of endophytic colonisation
of tree 2 by Dothiorella spp. declined in the upper inflorescence and pedicel tissue (Fruit
fill I1 in Fig. 1 and Table 3). The decline in detection of Dothiorella spp. corresponded to
an increase in the frequency of prophylactic sprays with copper oxychloride, the appearance
of visible residues of the fungicide on tissue samples, and an increase in the frequency of
detection of A . alternata (Table 4).
By contrast, endophytic colonisation by stem end rot pathogens was rarely detected in
samples from the Mutchilba site with a history of low stem end rot levels. In samples from
Mutchilba, colonisation of stem and inflorescence tissue by Dothiorella spp. was only
detected at the base of the second youngest growth flush (I1 base) at flowering, and at the
base of the youngest growth flush (I base) at harvest (late mature) at incidences of 40%
and 20% respectively.
For branches from Childers, plating onto PDA of larger tissue pieces (cf. small tissue
pieces) revealed a low to moderate incidence of both Dothiorella spp. and P. mangiferae
at several isolation points (including 20% to 40% of pedicels) (Table 5). Levels of Dothiorella
spp. and P . rnangiferae detected on samples from an unsprayed tree at Indooroopilly were
high at all isolation points, including 60% to 100% of pedicels. Subsequently, high levels

Table 4. Average sequential incidence of Alternaria alternata in various segments of the

mango stern, inflorescence and pedicel tissue of five branches per growth stage from an
orchard with a history of high stem end rot losses. Growth stages listed are as illustrated in
Fig. 1
Incidence (%) of Alternuria alternufa at different growth stages
Fruit fill Fruit fill Late
Site: Ayr Pre-flowering Flowering Fruitset Young fruit I I1 mature
Days after flowering: 0 14 42 57 74 112
Growth flush
V 0 - L - -
IV 0 - - - -
I11 0 0 - - -
I1 base - 0 - - -
mid 0 0 - -
top - 0 20 - 0
1 base - 0 20 - 0
mid 0 0 20 - 60
top 20 0 0 0 20
Inflorescence
base 20 0 0 60 40
low 20 20 0 40 40
mid 40 0 0 40 -
top 0 20 0 20 -
tip 0 -
Side inflorescence
base 40 0 0 0 20
low 0 0 0 0 0
mid 0 0 40 -
Pedicel 0 20 40 20
Pedicel-fruit - L - 40
Mean total detections* 3.3 0 12.7 5.0 6.7 24.0 24.0
* Average incidence to/.) of A . alternata over all isolation points within a sample.
- No isolations made.
 Endophytes cause mango stem end rot 231

Table 5. Average incidence of endophytic (i) Dothiorella spp. and (ii) Phomopsis mangiferae
in segments of the mango stem and inflorescence tissue of five branches per growth stage,
from orchards during harvest, as detected by plating out either (a) small or (b) large tissue
pieces. Growth stages are as listed in Fig. 1
Incidence (%) at
Childers Indooroopilly I1
Growth stage: Mature (a) Late Mature (b) Mature (b)
Growth flush (i) (ii) (9 (ii) (4 (ii)
IV 0 0 - - - -
I11 base - - 67 33 60 0
top 20 40 40 0 100 95
I1 base 0 20 40 40 40 80
top - - 20 60 75 75
I base 60 20 0 0 100 80
top - - 80 40 100 80
Inflorescence
base 0 20 20 40 100 100
mid - - - - - -
Side inflorescence 0 0 40 40 80 80
Peduncle 0 0 20 40 40 80
Pedicel 20 0 20 40 60 100
Mean total detections* 12.5 12.5 34.7 33.3 75.5 83.0
Stem end rot** 20 0 100 0 20 0
* Average incidence over all isolation points (from five branches) for the growth stage
sampled.
* * Incidence of stem end rot caused by Dothiorella spp. on harvested fruit after 21 days at
23°C.
- No isolations made.

Table 6. In vitro interactions of Dothiorella spp. and Alternaria alternata at uarious

temperatures after 21 days
Recovery % of Alternaria alone (A) or both Alternaria and Dothiorella (AD) after incubation at various
temperatures
A . Alternata * * with D . dominicana D. mangiferae D. 'long'
Incidence (%)
Temperature "C AD A AD A AD A
11 0 100 0 100 0 100
14.5 0 100 0 100 0 100
19 62.5 31.5 0 100 0 100
21 100 0 100 0 100 0
20-25 * 100 0 100 0 100 0
25 100 0 100 0 100 0
28 100 0 100 0 100 0
37 0 0 100 D.m.** 0 100 D.I.** 0
40 0 0 0 0 0 0
* With near-UV light on a 12 illumination cycle.
* * A . alternata failed to grow. Only D. mangiferae (D.m.) or D . 'long' (D.1). grew at this
temperature.
232 G. I. JOHNSON, A. J. MEAD, A. W. COOKE AND J . R. DEAN

of stem end rot (all fruit affected) developed in fruit from the late-mature stage branches
from Childers, while low levels occurred in fruit from the mature stage branches from
Indooroopilly samples (Table 5 ) , which also developed anthracnose (all fruit affected).
Laboratory studies on in vitro interactions between Dothiorella spp. and A . alternata
showed that for similar levels of inoculum under near-UV at 20-25"C, and in darkness at
25°C or 28"C, distinguishable growth sectors of each Dothiorella sp. and A . alternata
occurred on plates inoculated with mixed inoculum. At 18"C, only D . dominicana produced
separate colony sectors in the presence of A . alternata, while at 11°C and 14.5"C only growth
of A . alternata was observed on plates co-inoculated with any Dothiorella sp. Only D .
mangiferae and D . 'long' grew at 37°C (Table 6).
Most isolates of Dothiorella were usually identified to genus only. However, for the
branches from tree 1 at the young fruit stage, D . dominicana was isolated consistently from
some segments along one stem, while D . 'long' was isolated consistently from some segments
along another stem.

Discussion
We have shown that Dothiorella spp., P . mangiferae and other stem end rot fungi occur
as endophytes of mature mango stems. Colonisation of inflorescence tissue by Dothiorella
spp. and P . mangiferae extends to the pedicels of young fruit by 57 days after flowering
(Table 4). After this time, colonisation by (or detection of) Dothiorella spp. and P .
mangiferae on sprayed trees could have been hampered by the accumulation of copper
residues on inflorescence surface tissue, or competitive inhibition from A . alternata on
isolation plates. Increasing the size of tissue sections plated out appeared to overcome the
problem of low detection levels, suggesting that the fungi were present, but sparsely
distributed.
In vitro studies indicated that at 20-25"C, the presence of A . alternata in tissue should
not prevent the successful detection of Dothiorella spp. when mycelia of both are present
in similar quantities.
Peterson, Johnson, Schipke & Cooke (1991) observed some reduction in stem end rot of
mangoes by copper oxychloride field sprays applied between fruit set and harvest, supporting
our view that the build-up in copper fungicide residues on the stem tissue of our samples
reduced colonisation and viability, or interfered with the isolation of endophytic colonisers.
The high incidence of detection of endophytes in unsprayed trees from Indooroopilly in the
present study also implies a role for copper sprays in reducing detection of colonisation in
sprayed trees.
The consistent isolation of D . dominicana at various points along the length of one branch
sample, and D . 'long' at various points along another branch sample provides evidence that
endophytic colonisation results from the growth of hyphae from one fungus colony along a
branch, rather than from discrete colonies developing from propagules deposited at random
on that branch. In the latter case, various Dothiorella spp. would occur at random at any
point on one branch.
Our demonstration that stem end rot fungi are endophytic inhabitants of mango branches
opens the way for devising novel control methods for stem end rot. Crist & Schoeneweiss
(1975) and Pusey (1989) have shown that water stress and defoliation favour colonisation
of the stem tissue of temperate trees by the anamorph of Botryosphaeria dothidea (= D .
dominicana, G. I. Johnson, unpublished) causing stem canker and dieback. Foliage loss and
water stress could have similar effectson endophyte colonisation of the mango inflorescence.
Isolates from pedicel tissue of Childers samples detected only 20% of the fruit which
 Endophytes cause mango stem end rot 233

subsequently developed stem end rot, suggesting that up to 80% of stem end rot lesions
developed from other sources of infection. Our findings do not discount the possibility that
epiphytic growth by stem end rot fungi might also occur. However, endophytic growth
provides an avenue for colonisation without the constraints imposed by epiphytic com-
petition from other microorganisms, fungivores and environmental fluctations.
Despite high colonisation rates by P. mangiferae of branches from Indooroopilly and
Childers, the stem end rot lesions that developed were caused by Dothiorella spp. This
reflects the slower mycelial growth rate (Johnson et al., 1991a) in oitro and slower symptom
development in inoculated fruit of P. mangiferae (Brooks, 1941). Our results indicate that
a suite of endophytic stem end rot pathogens may be present in the stem end tissue of
mangoes at harvest. Pre-harvest factors, or subsequent storage conditions, may influence
which colonist gains the advantage enabling it to invade the fruit and cause stem end rot.
Peterson et al. (1991) noted changes in the spectrum of pathogens causing stem end rot of
mangoes depending upon the storage temperature.
In fruit affected by anthracnose (ex. Indooroopilly), only a low level of stem end rot
developed, despite extensive detection of Dothiorella spp. and P. mangiferae in the pedicel.
The quiescence of infections of C. gloeosporioides may break earlier during fruit ripening
than the quiescence of the stem end rot fungi in the pedicel. Also, developing anthracnose
lesions or other endophytic colonists of the pedicel could have suppressed the spread of
stem end rot fungi. Stem end rot fungi may be left ‘waiting in the wings’ when anthracnose
is severe. However once the latter is controlled, stem end rot may emerge as the major
cause of losses (Johnson et al., 1989).
Future work will examine cultural methods for control of stem end rot and competitive
interactions among stem end rot fungi and between stem end rot fungi and C. gloeo-
sporioides.

Acknowledgements
The work reported here was funded by the Australian Centre for International Agri-
cultural Research (ACIAR 8844). The authors thank Dr T. O’Hare, Mr T. Campbell and
Mr R. Holmes for assistance with sampling, Ms K. Cannon for technical assistance, Mrs
K. Hall for preparation of the manuscript, Mr W. Smith for artwork and Drs J. A. G. Irwin
and J . Pitt for useful discussions. The co-operation of mango producers in allowing access
to their properties for sampling is acknowledged.

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(Received 26 June 1991)