APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1995, p. 801–803 Vol. 61, No.

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0099-2240/95/$04.0010

Reliability of Poly B-411, a Polymeric Anthraquinone-Based Dye, in

Determining the Rot Type Caused by Wood-Inhabiting Fungi
L. J. COOKSON*
Division of Forest Products, Commonwealth Scientific and Industrial Research Organisation,
Rosebank MDC, Clayton, Victoria 3169, Australia
Received 31 August 1994/Accepted 26 November 1994

In both Basidiomycotina and Ascomycotina, Poly B-411 decolorization was an excellent indicator of the
ability to cause white rot: 109 of the 110 isolates of brown rot fungi tested definitely did not decolorize Poly
B-411, and 392 of the 401 mainly active isolates of white rot fungi decolorized Poly B-411. The Bavendamm
(tannic acid) reaction was a less reliable test: of 74 white rot isolates examined that could not decay wood, 6
decolorized Poly B-411, but 19 gave positive Bavendamm reactions. Of 80 isolates of Ascomycotina and
Deuteromycotina that do not cause white rot, only 4 decolorized Poly B-411, but 17 gave a positive Bavendamm
test.

The Basidiomycotina vary in the ability to decay the various
components of wood. Brown rot fungi degrade carbohydrates,
leaving most of the polyaromatic lignin intact, whereas white
rot fungi degrade both lignin and carbohydrate, so that rela-
tively more white cellulose fiber remains in rotted wood. The
Bavendamm reaction, involving the oxidation of aromatic acids
such as tannic acid and gallic acid to various brown quinones,
is the established procedure used for determining the rot type
of basidiomycete fungi (1, 2, 9). The Bavendamm reaction
detects the phenol oxidase laccase, which is part of the lignin-
degrading system of some fungi (14, 18). However, this method
fails to detect the ability of other fungi such as Phanerochaete
chrysosporium Burds (22) to cause white rot. Also, some As-
comycotina and Deuteromycotina can give a positive Baven-
damm reaction even though they do not produce white rot
(22). Poly B-411 has been shown to be useful in studies on
lignin biodegradation (4, 6–8, 11–13, 20, 21, 24), because both
lignin and the dye are aromatic and polymeric in nature. Poly
B-411 is a blue, anthraquinone-based dye (12), the synthesis of
which was described by Dawson (10). The culture collection of
wood-inhabiting fungi, held at the Division of Forest Products
(15), contains many named cultures of known rot type. From
this collection, 662 isolates were examined for the ability to
degrade Poly B-411. Of these isolates, 249 were also examined
for the ability to oxidize tannic acid and produce the Baven-
damm reaction.
Poly B-411 plates were prepared by mixing 15 g of malt
extract (Oxoid), 10 g of no. 1 agar (Oxoid), and 0.2 g of Poly
B-411 (Sigma [Poly B-411 is a trademark of Dynapol]) per liter
of deionized water. The medium was autoclaved for 30 min,
and 20 ml was added to each plastic petri dish. Fungi were
subcultured from oil vials onto malt agar plates, and the plates
were used within 3 weeks. Each plate was inoculated centrally
with a 4-mm-long cube plug (colonized side up) and incubated
at 268C. There was one or more replicate for each isolate.
Some isolates were tested again after further subculturing on
malt agar plates. Poly B-411 plates were generally examined 4,
10, and 24 days after inoculation.
A selection of the isolates, including those isolates that grew
quickly on plates, were also incubated at 398C on Poly B-411
* Mailing address: CSIRO, Division of Forest Products, Private Bag
10, Rosebank MDC, Clayton, Victoria 3169, Australia.
plates and at 268C on tannic acid plates. Tannic acid plates
were prepared by adding 15 g of malt extract, 10 g of no. 1 agar,
and 1 g of tannic acid to each liter of deionized water. The
tannic acid was autoclaved in 100 ml of water, in a flask sep-
arate from the 900 ml of malt agar, and the two solutions were
mixed before being poured. Another selection of isolates, in-
cluding those that did not give the expected reactions on Poly
B-411 plates, were grown on poplar feeder strips in soil jars
with a sapwood block of Pinus radiata D. Don and Eucalyptus
obliqua L’Herit., to confirm rot type. The soil jars were similar
to those described by Cookson and Greaves (5). Jars were
placed in trays, along with a jar of water for humidity. Each
tray was encased in a plastic bag, and incubation was at 268C
for 12 weeks. Jars were opened in a sterile air cabinet, and for
those fungi that had grown successfully, hyphal scrapings taken
from blocks were used to inoculate another set of Poly B-411
plates. These reisolated fungi were incubated for 2 to 3 weeks
to determine their reaction to Poly B-411. After the hyphal
scrapings were taken, rot type on blocks and feeder strips was
recorded if apparent.
For most fungi, there was little difference in the growth rate
obtained on malt agar with or without Poly B-411. However,
0.1% tannic acid in the malt agar reduced the growth rate of
most fungi and prevented growth by 20 of the 199 isolates of
the white rot fungi examined on both media.
The positive color reaction on Poly B-411 plates was gener-
ally easy to determine. Blue agar gradually turned pink and
then pale yellow (color of malt agar) if dye degradation was
complete. The pink coloration suggests partial degradation of
Poly B-411 to its amino-containing anthraquinone precursor,
which is red (10). None of the fungi produced a pigment that
could have confused interpretation of results with Poly B-411.
However, on tannic acid plates, some fungi produced yellow or
brown diffusate, which could be confused with the yellow-
brown corona of a positive Bavendamm reaction. The same
fungi on Poly B-411 plates turned agar green or blue-green
because of the mixing of blue dye with yellow-brown diffusate.
This green coloration appeared for 4% of the 662 isolates
examined and was interpreted as a negative reaction. If fungi
producing a yellow-brown diffusate could also degrade Poly
B-411, it was generally possible to detect a pink region on the
plates before the color was dominated by the fungal diffusate.

801
802 NOTES
TABLE 1. Reactions of isolates of Basidiomycotina, Ascomycotina,
and Deuteromycotina to Poly B-411 and tannic acid
No. of isolates
Plate type and fungi
Total Positive Negative
Poly B-411
White rot Basidiomycotina 469 392 77a
Brown rot Basidiomycotina 110 1 109
Ascomycotina and Deuteromycotina 83 7b 76
Tannic acid
White rot Basidiomycotina 199 155 44c
Brown rot Basidiomycotina 15 0 15
Ascomycotina and Deuteromycotina 35 20b 15
a
Nine isolates decayed wood, and 68 could not decay wood.
b
Three isolates could produce white rot in wood.
c
Eleven isolates could decay wood.
Also, if blue dye was degraded, then the agar would not turn
green.
The positive Poly B-411 reaction generally appeared 4 to 5
days after inoculation, whereas the Bavendamm reaction gen-
erally occurred within 3 days of inoculation. However, some
white rot fungi did not produce a positive Poly B-411 reaction
until 20 or more days’ growth. Fungal growth would have
reduced the available nitrogen present in the medium during
this time. Others (3, 16, 23) have reported that lignin break-
down usually intensifies with decreasing nitrogen levels, but
the threshold level of nitrogen required would vary between
fungi.
Of the isolates from the Division of Forest Products collec-
tion examined with Poly B-411, 110 representing 49 species of
Basidiomycotina were recorded as species that produce brown
rot. From this group, 109 isolates gave the negative Poly B-411
reactions expected of brown rots (Table 1). The only isolate to
give conflicting results was one of three Grifola campyla
(Berk.) isolates. It turned agar slightly pink on three separate
occasions, even though its rot type was confirmed by the soil jar
test to be brown. However, when plates were more than 24
days old, the agar became blue, confirming that the positive
reaction by this isolate was not strong. Therefore, Poly B-411
gave the correct rot type for more than 99% of the brown rots
examined.
Of the 469 isolates of white rot Basidiomycotina examined,
392 isolates, representing 156 species, gave positive Poly B-411
reactions. Of those isolates giving a positive reaction, 102 were
examined in the soil jar test, and 80 could cause white rot. The
remaining 22 isolates failed to produce decay, including 7 that
had given weak positive reactions. Only 6 of these 22 isolates
were also examined on tannic acid plates, and all gave a pos-
itive Bavendamm reaction.
Twenty isolates of white rot Basidiomycotina gave positive
Poly B-411 reactions after an initial negative reaction. The
positive reaction for 18 of these 20 isolates came after repeated
subculturing onto fresh malt agar plates, whereas it came after
reisolation from the surfaces of wooden blocks in soil jars for
the remaining two isolates.
Temperature also had some bearing on Poly B-411 reac-
tions. Of 162 white rot Basidiomycotina isolates incubated at
398C, 128 isolates failed to grow more than 20-mm diameter
after 22 days’ incubation and either failed to give a reaction or
any faint initial reaction disappeared within 14 days as agar
became uniformly blue once more. This temperature was cho-
sen, as it is the temperature generally used for studies with P.
chrysosporium (16). Of the 34 isolates able to grow adequately
APPL. ENVIRON. MICROBIOL.
at 398C, two isolates, Schizopora paradoxa (Schrader: Fr.)
Donk and a Poria sp., could not decolorize Poly B-411 at 268C
but could at 398C. Two other isolates, a member of the Hyd-
naceae and Corticium leptospermi G. Cunn., could decolorize
dye at 268C but not at 398C, whereas another four isolates
produced less complete decolorization of Poly B-411 at the
higher temperature than at the lower temperature.
Seventy-seven isolates of white rot fungi gave negative reac-
tions to Poly B-411. Sixty-eight of these also failed to cause
decay in wooden blocks in the soil jar test, even though 13 gave
positive Bavendamm reactions. The negative reaction to Poly
B-411 therefore might be due to a loss in vitality or an inability
to cause decay and decolorization under the culture conditions
in which they were tested. These isolates were not considered
to represent a failure of the dye to determine rot capacity
under the test conditions used. Some loss in vitality is sug-
gested in that 65% of these isolates came from the oldest half
of the collection, while the remaining 35% of isolates were
more recently collected.
The species that failed to be detected as white rot fungi by
Poly B-411 but were able to produce white rot in the soil jar
test, included the white pocket rots Xylobolus frustulatus (Pers.:
Fr.) Boidin and Xylobolus illudens (Berk.) Boidin. Both species
also failed to give a Bavendamm reaction, which was reported
by Wang and Zabel (25) to be a characteristic of X. frustulatus.
Hymenochaete tasmanica Massee also failed to degrade Poly
B-411, even though it produced white rot in the soil jar test.
Unlike the two Xylobolus species, H. tasmanica gave a positive
Bavendamm reaction. Also, one of six isolates of Phellinus
robustus (Karst.) Bourdot & Galzin failed to give a positive
Poly B-411 reaction on two separate occasions, even though it
could produce a small amount of white rot in E. obliqua blocks.
Therefore, of the 469 isolates of white rot fungi examined, only
392 isolates (83.6%) were correctly determined to be white rot
fungi by Poly B-411. However, while 68 isolates (14.5%) failed
to give a positive reaction, they also failed to produce decay in
soil jars. Only nine isolates (1.9%) of white rot fungi gave
negative Poly B-411 reactions while also being able to actively
produce white rot.
Inconsistent results with Poly B-411 were found among iso-
lates of the genus Hymenochaete and Lopharia crassa (Lev.)
Boidin. For Hymenochaete spp., 15 isolates from eight species
were examined. All were expected to react as white rot fungi
on the basis of published rot types of the species involved and
the rot type of the wood from which the isolates were originally
taken. However, only five isolates gave positive Poly B-411
reactions, while these plus another five gave positive Baven-
damm reactions. However, only three of the Hymenochaete
isolates were able to produce decay. Similarly for L. crassa,
eight isolates were examined and all gave a positive Baven-
damm reaction, whereas only four gave a positive Poly B-411
reaction. However, there were only four isolates that could
produce white rot in the decay test, and these were the isolates
that tested positive against Poly B-411.
Eighty-three isolates of Ascomycotina and Deuteromycotina
were examined, representing about 58 different species.
Twenty of these isolates gave a positive Bavendamm reaction,
whereas only seven gave a positive Poly B-411 reaction. Those
isolates that gave a positive Bavendamm reaction but were not
able to degrade Poly B-411 were Daldinia concentrica (Bolton)
Ces & de Not., Ceratocystis sp., Hypoxylon hypomiltum Mont.,
and nine Xylaria isolates. Those isolates able both to give a
Bavendamm reaction and to degrade Poly B-411 were Alter-
naria sp., Aurophora dochmia (Berk. & Curt.) Rifai, Hypoxylon
sp., Xylaria anisopleura (Mont.) Dennis, and Xylaria sp. Of the
Ascomycotina and Deuteromycotina examined, only the Hy-
VOL. 61, 1995
poxylon isolate and both X. anisopleura isolates listed in the
latter group could produce white rot in the soil jar test. The
ability of some Ascomycotina such as some Hypoxylon and
Xylaria spp. to cause decay similar to white rot was noted by
Kirk (17).
The results show that Poly B-411 could readily determine,
from a large number of isolates, those fungi able to actively
produce white rot. The Bavendamm method was less useful in
this regard. Tannic acid reacts to laccase especially (14), while
Poly B-411 is little affected (4). However, several other en-
zymes seem more important in determining the ability to de-
grade lignin. For example, P. chrysosporium produces no de-
tectable laccase (18). Poly B-411 may require the more
complete set of lignin-degrading enzymes, such as manganese
peroxidase (6, 11) and lignin peroxidase (6, 19), before it can
be degraded.
I thank both Andrew Martin and Gary Johnson for the work they did
in subculturing and maintaining the culture collection and for their
comments on the manuscript. Thanks also to Barry Macauley for his
comments on the manuscript.
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