Further investigations on the biology of Phomopsis cinerascens, the cause of fig canker in Iran

Abstract

Fig branch canker is a major disease in most parts of Iran, especially in Estahban (Fars province), which has the largest area of dry fig plantations in that country. In 1999-2000 a general survey was conducted in rainfed fig plantations throughout Fars province. In this survey Phomopsis cinerascens was consistently isolated from the cankers. The fungus produced pycnidia containing a-conidia on active cankers from fall to mid spring. No β-conidia were found under natural conditions, but many isolates produced β-conidia intermixed with α-conidia in culture. Only α-conidia germinated on agar medium. The optimum temperature for growth, pycnidial formation and pycnidiospore germination was 25°C. Pathogenicity tests revealed that the fungus infected inoculated branches at 15-25°C but no infection occurred at 5°C or at 30°C or higher. Under field conditions, the pathogen infected branches from fall to mid spring, but little infection occurred in summer. The pruning wounds remained receptive to the pathogen from fall to mid spring. Pycnidiospores that over-summered on trees or on branches lying on orchard floors were not viable. Infected branches under moist conditions produced new pycnidia containing viable conidia. Mycelia are considered important for over-summering the pathogen in Fars province.

Full text

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Corresponding author: Z. Banihashemi
Fax: +98 711 2294818
E-mail: ziabani@shirazu.ac.ir
Introduction
The largest areas of rainfed caprifig (Ficus carica
L.) used for production of dry figs in Iran are located in
Estahban in Fars province. More than 20,000 ha of dry figs
are grown in this province, with annual production of over
20,000 tonnes (Javadi, 2001).
In 1978 fig branch canker was detected in Estahban.
The causal agent was identified as Phomopsis sp. (Zia
Banihashemi, unpublished data) and control measures were
initiated (Fatemi and Mobayyan, 1983). Further surveys
indicated that the pathogen occurred in most rainfed fig
plantations in Fars Province (Javadi, 2001). The disease was
originally reported from Italy in 1878 by Saccardo and the
causal organism was identified as Phomopsis cinerascens
(teleomorph: Diaporthe cinerascens) by Grove in 1935
(cited by Ogawa and English, 1990). It caused a major
epidemic on the cultivar Kadota in California, due to heavy
pruning (Ferguson et al., 1990). The pathogen affects all
commercial figs in California (Ogawa and English, 1991).
It survives from one year to the next in cankers on the trees
or on infected branches in orchards (Hansen, 1949). After
infection, conidia are produced in the cankers during the wet
period in winter (English, 1951). Rain splash and pruning
tools are the main means whereby the pathogen is dispersed
(Hansen, 1949). In California susceptible fig cultivars such
as Kadota are prone to infection from November through
February, but become resistant thereafter (English, 1951;
1952b). The optimum temperature for growth of the
pathogen in culture is 25°C, and isolates do not grow at 4
or 30°C or higher (Ogawa and English, 1991).
The objective of the present study was to investigate
the biology of the pathogen concentrating on its mode
Key words: Ficus carica, Phomopsis canker, rainfed g, survival.
Summary. Fig branch canker is a major disease in most parts of Iran, especially in Estahban (Fars province), which
has the largest area of dry g plantations in that country. In 1999–2000 a general survey was conducted in rainfed
g plantations throughout Fars province. In this survey Phomopsis cinerascens was consistently isolated from the
cankers. The fungus produced pycnidia containing α-conidia on active cankers from fall to mid spring. No β-conidia
were found under natural conditions, but many isolates produced β-conidia intermixed with α-conidia in culture. Only
α-conidia germinated on agar medium. The optimum temperature for growth, pycnidial formation and pycnidiospore
germination was 25°C. Pathogenicity tests revealed that the fungus infected inoculated branches at 15–25°C but no
infection occurred at 5°C or at 30°C or higher. Under eld conditions, the pathogen infected branches from fall to mid
spring, but little infection occurred in summer. The pruning wounds remained receptive to the pathogen from fall
to mid spring. Pycnidiospores that over-summered on trees or on branches lying on orchard oors were not viable.
Infected branches under moist conditions produced new pycnidia containing viable conidia. Mycelia are considered
important for over-summering the pathogen in Fars province.
Zia BaNiHaSHEMi and ali REZa JaVaDi
Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz, Iran
Further investigations on the biology of Phomopsis cinerascens,
the cause of g canker in Iran
Phytopathol. Mediterr. (2009) 48, 454–460

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Biology of Phomopsis cinerascens in Iran
of survival and on the receptiveness of pruning wounds
grown in rainfed dry fig plantations in Estahban. Part of this
research has been reported earlier (Javadi and Banihashemi,
2005).
Materials and methods
Sample collection and isolation
During 1999–2000 a number of fig plantations in
Estahban and other locations in Fars province were visited
and symptomatic branches showing wilting or canker were
collected and taken to the laboratory. The causal agent was
isolated from samples collected from November to May.
Diseased tissue on each branch was washed with tap water
and the outer bark was carefully removed with a scalpel
to expose the layer underlying the discolored tissue. From
the margin of healthy and discolored tissues, a few wood
fragments (0.5–1×0.2 cm) were excised, surface-sterilized
in 0.5% sodium hypochlorite for 1–3 minutes, rinsed with
sterile distilled water (SDW), blotted dry with a sterile paper
towel, placed on Petri dishes containing potato dextrose
agar (PDA) and incubated at 25°C. After 1 week, fungal
colonies appeared around each fragment. However, with
samples collected after May and throughout the summer,
the above isolation method was not suitable. Cankers with
pycnidia not older than 3 months were surface-sterilized
with 95% ethanol and the pycnidia were removed with
a sterile scalpel, transferred to a test tube containing
SDW and vortexed. The suspension containing α-conidia
was streaked on 2% water agar (WA) in Petri plates and
incubated at 25°C. After 24 h, germinated conidia were
transferred to PDA plates. If pycnidia were not present upon
sampling, the infected branches were surface-sterilized in
0.5% sodium hypochlorite for 5 min., placed in sterilized
glass jars contained moist sponge, and incubated at 25°C.
After 12–15 days the pycnidia were formed and exuded
pycnidiospores as cirri, which were transferred to test tubes
containing SDW, vortexed and streaked on 2% WA plates
as described above. All cultures for further study were from
single conidia, and these were stored at 4°C.
Wood colonization tests in the laboratory
Healthy fig bran ches 1–2 cm in diameter were
collected from the fig plantation and taken immediately to
the laboratory. They were cut into 15–20 cm portions and
dipped into 0.5% sodium hypochlorite for 5 min., blotted
dry with a paper towel and the cut ends were dipped in
melted paraffin wax (70°C) to reduce water loss. The middle
portion of each branch was surface-sterilized with 95%
ethyl alcohol. Three cuts were made in the bark on each
side of a square. A block of agar 8 mm in diameter and 2
mm thick colonized by the pathogen was inserted under the
bark, covered with the bark and wrapped with Parafilm to
reduce desiccation. Experimental controls were inoculated
with uncolonized PDA in a similar manner. Inoculated
branches were stored in sterilized glass jars containing a
moist sponge and incubated at 20–25°C for 10–15 days
until the disease symptoms appeared.
Pathogenicity test in the orchard
A few fig trees from one local cultivar and of the
same age were selected. From each tree a few branches,
2–3 cm in diameter were inoculated in situ as described
above. After 12–20 days, the branches were cut from the
trees and transferred to the laboratory to check for disease
development and to re-isolate the pathogen.
Cardinal temperature for growth and germination
Several isolates of P. cinerascens from different
locations were transferred to Petri dishes containing
PDA and incubated at 15, 20, 25, 30 or 35°C. Five
dishes were used for each combination of isolate and
temperature. Colony diameters were measured after
12 days. Pycnidiospore suspensions (103 conidia mL-1)
obtained from PDA cultures were streaked on 2% WA and
incubated at different temperatures between 5 and 35°C
(5°C increments). Five replications were used for each
temperature. Conidial germination and germ tube length
were measured after 24 h.
Effect of temperature on disease development
Detached fig branches were inoculated as described
above and incubated at 15, 20, 25, 30 or 35°C in jars
containing moist sponges. Ten branches were used at each
temperature. After 15 days, infection was assessed by re-
isolation of the pathogen from beyond each inoculated
point.
Effect of temperature on pycnidium formation
Several fig branches were inoculated as described
above and kept at room temperature for 10 days before
they were incubated at different temperatures between
5 and 35°C (5°C increments). Branches were left until
the pycnidia appeared. Ten branches were used at each
temperature.
Duration of g susceptibility to infection
To investigate infection and canker development at
different times of the year, ten similar aged fig trees of one
local cultivar were selected at the Estahban Fig Research

Phytopathologia Mediterranea
Z. Banihashemi and A.R. Javadi
456
Station. At the beginning of each month, four branches
(2.5–3 cm diameter) from each tree were inoculated as
described for detached branches (two branches with the
pathogen and two with PDA). At the end of each month, the
branches were cut and transferred to the laboratory where
the length of the canker on each branch was measured and
the pathogen was reisolated. The experiment was repeated
each month throughout 1 year.
Receptiveness of pruning wounds to infection
Ten fig trees cv. Sabz of the same age and size were
selected. In early December, about 140 branches (diameter
of 1.5 cm) were pruned. Every 30 days the pruning wounds
of ten branches were inoculated with the pathogen and two
branches with sterile PDA as experimental controls. The
wounds were then sealed with Parafilm. After 30 days, the
inoculated branches were removed and transferred to the
laboratory for the re-isolation of the pathogen from beyond
the point of inoculation.
Survival of the pathogen
At different seasons, fig plantations throughout the
province were inspected several times and pruned branches
left on the floor of each plantation were collected, washed
and wrapped in wet cloth, placed in a nylon bag and left in
the orchard. After 2–3 weeks, a few branches with pycnidia
were brought to the laboratory and the germinability of the
pycnidiospores was determined.
Host range study
Detached and intact branches of the woody plants
grapevine (Vitis vinifera L.), apple (Malus communis Desf.),
pear (Pyrus communis L.), pistachio, (Pistacia vera L.),
apricot (Prunus armeniaca L.), peach (Prunus persica
Stokes), sweet orange (Citrus sinensis Otbeck), walnut
(Juglans regia L.), mulberry (Morus alba L.) and plane tree
(Platanus orientalis L.) were inoculated with P. cinerascens
using the method described above. Results were recorded
after 15 days in the laboratory and 25 days in the field.
Results
Isolation and identication
Forty isolates of the pathogen were recovered from
cankers collected from different parts of the province. All
isolates produced pycnidia on PDA. On fig branches the
pycnidia were aggregated, immersed in the bark, globose-
depressed, 180–450 µm in diameter, bi- or uni-loculate
with the ostiole emerging through the surface of the canker
(Figure 1).
Most pycnidia contained α-conidia, which were hyaline,
elliptic-fusoid, 7–10×2.5–4 μm, and often biguttulate. No
β-conidia were detected in pycnidia collected from the
branches. On PDA, however, several pathogen isolates
collected from different parts of the province produced
pycnidia (Figure 2) containing β-conidia intermixed with
α-conidia (Figure 3). They were filiform, mostly hooked,
1×14–25 μm. Only α-conidia germinated on the agar
medium, while β-conidia never germinated. No sexual
stage of the fungus was found under natural or laboratory
conditions. The optimum temperature for growth and
Figure 1. Cross-section through a pycnidium of Phomopsis
cinerascens on a g branch (scale bar = 45 µm).
Figure 2. Formation of pycnidium of Phomopsis
cinerascens on PDA after 4 weeks incubation at 25°C and
production of cirri with α and β-conidia.

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Biology of Phomopsis cinerascens in Iran
Figure 4. Effect of temperature on colony diameter of
Phomopsis cinerascens on PDA after 14 days incubation
in the dark. Columns followed by the same letter are not
signicantly different at P<0.01 according to Duncan’s
multiple range test.
Figure 5. Percentage germination of α conidia of
Phomopsis cinerascens after 24 hours on water agar at
different temperatures. Columns followed by the same
letter are not signicantly different at P<0.01 according
to Duncan’s multiple range test.
germination was 25°C. No growth or germination occurred
at or below 5°C. None of the woody plant species (other than
fig) that were inoculated with the fungus became infected
by the pathogen. Based on morphological and cultural
features and on host specificity the fungus was identified
as Phomopsis cinerascens.
Temperature effects
The optimum temperature for growth was 25°C (range
15–30°C) with no growth occurring at 35°C (Figure 4). The
optimum temperature for conidial germination and germ
tube elongation was 25°C (Figures 5 and 6). Although
conidial germination was high between 20 and 30°C, germ
tube elongation was reduced below 20 and above 30°C.
Figure 6. Germ tube length of α conidia of Phomopsis
cinerascens after 24 hours of incubation at different
temperatures. Columns followed by the same letter are
not signicantly different at P<0.01 according to Duncan’s
multiple range test.
The pathogen infected detached fig branches at 15–
25°C but no infection occurred at 5 or 30°C or above.
Pycnidia formed on infected branches between 5 and 25°C,
but the time of incubation for pycnidium formation was longer
(17–22 days) at 5°C than at 20 or 25°C (4–7 days).
Duration of g tree susceptibility to the infection
There were statistically significant differences (P≤0.01)
in canker development between dates of inoculation. The
infection rate was low in July and August. From fall to early
spring the trees were susceptible to infection, after which
time susceptibility declined (Figure 7). The weather data
indicated that temperature was more critical than humidity
as a factor affecting infection and disease development.
Figure 3. Mixture of α and β-conidia of Phomopsis
cinerascens (scale bar = 16 µm).

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Receptiveness of pruning wound to infection
Pruning wounds made in December remained receptive
to the pathogen throughout the year but they were most
receptive from fall to mid spring (Figure 8).
Survival of the pathogen
Infected branches collected in the fall, winter and
early spring produced pycnidia after they were incubated
for 2–3 weeks under humid conditions. No pycnidia were
seen on the branches collected in summer; these branches
were colonized by saprophytic fungi. The old pycnidia left
on the branches from before the fall contained non-viable
conidia.
Host range study
Inoculated and non-inoculated, attached and detached
branches of different plant species were inspected 15 and
25 days after inoculation. Only inoculated fig branches
developed cankers and produced pycnidia containing
pycnidiospores of P. cinerascens, and only from these
branches was the pathogen re-isolated. There was limited
infection on mulberry but the pathogen did not spread
beyond the point of inoculation. No symptoms developed
Figure 7. Relationship between the mean temperature and Phomopsis cinerascens canker development on g trees
during 2000–2001 under Estahban (Fars, Iran) conditions. Columns followed by the same letters are not signicantly
different at P<0.01 according to Duncan’s multiple range test.
on any of the other inoculated hosts, and the pathogen was
not re-isolated from these inoculated plants.
Discussion
This is the first comprehensive study of P. cinerascens in
rainfed fig plantations in Iran. The pathogen was active during
the dormant period of the host, but when growth resumed
the host resisted infection. Similar results were reported in
California (English, 1951, 1952a, 1952b, 1962). Kadota
fig trees in Californian orchards were immune to infection
from April and through the growing season, but were highly
susceptible from November to February. New pruning cuts
were susceptible to infection when moisture and temperature
conditions were favourable (English 1952, 1958). The present
study found that pruning cuts under Estahban conditions
remained receptive to the pathogen in fall, winter and early
spring but became resistant thereafter.
Phomopsis cinerascens is a wound invader: the
pathogen invades host branches mainly through pruning
cuts, but sunburn and frost probably also enable infection.
English (1951) also suggested that wounds and bark killed
by frost and sunburn were the chief avenues of infection,

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Biology of Phomopsis cinerascens in Iran
Figure 8. Duration of pruning wound receptiveness to Phomopsis cinerascens on g trees from November to June.
Columns followed by the same letter are not signicantly different at P≤0.01 according to Duncan multiple range
test.
and that leaf scars were only of minor importance.
Fig branches were susceptible to infection in fall, winter
and early spring, but branches were not susceptible in July
and August. The lack of rapid canker spread from April to
October has been assumed to be due to the active growth
of the host (Ogawa and English, 1991).
Under laboratory conditions, the optimum temperature
for growth and spore germination of P. cinerascens was
25°C and no infection occurred at 5oC or below or 30°C or
above. A suitable temperature seemed to be more critical
than moisture. The weather data in Estahban over the last
13 years indicated that temperature plays an important role
in canker development.
In the present study it was found that oversummered
pycnidiospores of P. cinerascens we re not viable
on can ke rs rec ov er ed fro m ca nk er-affected field-
grown branches, so we assume that mycelium is the
main survival agent. With appropriate humidity and
temperature, branches with oversummered pycnidia on
trees and on the orchard floors produce new pycnidia
with viable conidia. These conidia disseminate the
pathogen and cause new infections when temperature
and moisture conditions are favourable.
Pycnidia collected under natural conditions contained
only α-conidia, which are capable of germination. Under
laboratory conditions some isolates on PDA produced β-
conidia intermixed with α-conidia, but these β-conidia did
not germinate on any of the media examined in this study.
Uddin et al. (1995) reported that Phomopsis sp., which
causes peach Phomopsis canker, did not produce β-conidia
on some media and under certain environmental conditions.
The role of β-conidia in the epidemiology of these diseases
is therefore unclear.
Since pruning cuts remain receptive to P. cinerascens
for long periods, the best strategy to manage the disease
would be to delay pruning as much as possible. However,
late pruning has been reported to adversely affect fruit
maturity (English, 1953). Uddin and Stevenson (1998)
suggested that in peach late pruning may reduce infection by
Phomopsis sp. The effect of late pruning of rainfed caprifig
in Estahban has not been investigated.
Acknowledgements
The authors are grateful to the Shiraz University
Research Council for financial support (project number
79-AG-1344-211).

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Accepted for publication: August 31, 2007