J. Gen. Plant Pathol. 67 : 175-181 (2001)

A Root Box Method to Estimate Virulence of He/icobasidium mompa Using Carrots and Its Comparison with the Conventional Method Using Apple Stocks

Yukari UETAKE1,2, Hitoshi NAKAMURA', Masao ARAKAWA', lkuko OKABE3 and Naoyuki MATSUMOT03*

ABSTRACT

A root box method with carrots was developed to estimate virulence of the violet root rot fungus, Helicobasidium mompa, to facilitate short-term screening of many isolates during a year. The root box consisted of two transparent acrylic plates and a plastic bag of vermiculite in which two taproots of carrot were growing and inoculated with the fungus growing on fragments of mulberry twigs. The boxes were kept in a greenhouse at 25C, and the surface of carrots was observed weekly up to 14 weeks. The virulence of each isolate was determined based on the number of weeks after inoculation required for the fungus to develop infection cushions on the surface of carrots. Results were compared with those from the conventional inoculation method using apple stocks. Two-year-old 456 apple stocks were planted with or without fungal inoculum in 30-cm diam. plastic pots containing commercial soil and placed outdoors in April 1999. Symptoms on plant tops were observed weekly, and the first stocks were killed 14 weeks after inoculation. At the end of trial 1 (6 months) and trial 2 (14 months), apple stocks were dug up to rate disease index (DI) based on hyphal growth and infection cushion formation on the stem base. There was variability in disease severity among replicates as well as isolate variability ; however, the results were similar in both trails. The level of virulence estimated by both methods was almost parallel for a total of 23 isolates from five plant species, except for two isolates from sweet potato that formed no obvious infection cushion on apple roots but on carrot were the most virulent.

(Received December 11, 2000 ; Accepted March 23, 2001)

Key words : inoculation method, Helicobasidium mompa, violet root rot, virulence.

INTRODUCTION

Violet root rot caused by a basidiomycetous fungus, Helicobasidium mompa Tanaka, causes serious damage on apple trees in Japan. The fungus attacks a wide range of plants, and 104 plant species belonging to 44 families have been identified as hosts7). The fungus spreads on the root surface as mycelial strands, which ultimately de- velop into hyphal mats. Infection cushions, produced un- derneath the mats, infect the host t i ~ s u e s ~ - ~ , ' ~ ) . Control of violet root rot is difficult both chemically and biologically.

Matsumoto') proposed exploiting dsRNA to control root diseases of fruit trees. Screening less virulent isolates is essential to obtain hypovirulent isolates ; however, few

reports on inoculation tests are available for the violet root rot fungus. Although most researches in Japan inocu- late apple stocks in the field') (referred to as conventional method), this method limits the number of replicates and is time-consuming. A few greenhouse or laboratory methods have been reported using various plants, such as alfalfag), apple trees9), asparagusg), carrots9), cucumber'0), sweet potato7) and potato4). They were not intended for determining isolate variability in virulence or for compar- ing results with those from the conventional method.

The purpose of this study was to develop a greenhouse method to minimize the incubation period for inoculated plants and to reduce space, allowing screening of many isolates throughout the year. A root box method") was developed for carrots, which are highly susceptible to the

I Bio-oriented Technology Research Advancement Institution, National Institute for Ago-Environmental Sciences, Tsukuba 305-8604, Japan Present address : Bio-oriented Technology Research Advancement Institution, National Institute of Livestock and Grassland Science, Nishinasuno 329-2793, Japan National Institute for Ago-Environmental Sciences, Tsukuba 305-8604, Japan

* Corresponding author (E-mail : nowmat @ affrc.go.jp)

176 JGPP

d i ~ e a s e ' ~ ) and can be produced all year in a greenhouse. The results from this root box method were compared with those of the conventional field method.

MATERIALS AND METHODS

Conventional method Twenty-three isolates of the violet root rot fungus from five hosts from all over Japan were selected (Table 1). Mulberry twigs (ca. 7-10 mm diam.) were cut into pieces (1.5-2 cm long) and soaked overnight in water. Fifty g of these fragments were put in a plastic jar (60X60X100mm ; IWAKI, Funabashi), and autoclaved at 121°C for 40 min. A piece (7cm') of oatmeal agar (OMA, Difco, Detroit) culture was removed from 9-cm diam. plates, placed in the jar among the twig fragments, and incubated for 3 months at 25°C. Autoclaved mulberry twigs were used as controls.

Roots of apple stocks (JM7, 2 years old) were folded over a mass of fungal inoculum that was removed care- fully from the plastic jar, then planted in a plastic pot (30 cm diam., 30 cm height) of commercial soil (Kanuma soil). The pots were buried at random in the field. The experiments consisted of trials 1 and 2 with 9 and 10 replications, respectively, starting a t the beginning of April 1999. Trees were trimmed to 50cm tall and watered just after treatment.

Table 1. Isolates of Helicobasidium mompa used in this study

Isolate Host Locality 1 2 3 4 5 6

10 11 12 13 14 17 18 21 26 32 35 38 48 70

136 169 189

Fukushima Fukushima Fukushima Fukushima Fukushima Fukushima Fukushima Fukushima Fukushima Fukushima Kumamoto Saitama Hokkaido Akita Akita Akita Akita Akita Akita Fukushima Kumamoto Iwate Ibaraki

Symptoms on plant tops were checked weekly, and wilted plants were dug up to examine the surface of the stem base under a dissecting microscope. All plants were dug up at the end of each trial, i.e., at the beginning of October 1999 for trial 1 and at the beginning of June 2000 for trial 2. Because roots were often decomposed and detached from the stem when plants were severely diseased, disease index (DI) was rated on the basis of signs on the stem base of the stocks : DI 0, no signs ; DI 1, with hyphae and/or mycelial strands ; DI 1.5, mycelial mat without infection cushions ; DI 2, infection cushions (Fig. 1A-C) ; and DI 3, plants wilted and killed before the end of trials.

The fungus was pre-grown on OMA dispensed into thick Petri dishes (9 cm diam., 2 cm thick) for 3 weeks at 25°C. Twelve autoclaved fragments of mulberry twigs prepared as before were subsequently placed directly on the fungal colony and incubated further for 3 weeks at 25°C. The root box method devised by Suzui") was modified to screen weakly virulent isolates.

Root box method

P

- - 'W

Fig. 1. Infection cushions on underground parts of apple plant inoculated by the conventional method (A- C). A, side appearance of an infection cushion (arrow) with a peg underneath (arrowhead) after removal from stem bark with a needle. A hole (double arrowhead) remained in the bark at the place of the infection peg. B, degrading infection cushion (arrow). C, trace of an infection cushion after degradation (arrow). D, Mycelial mat formed on the carrot surface in a root box. Sclerotia (arrows) are formed beside the carrot, and hyphal strands (arrowhead) are extending into vermiculite.

Estimating Virulence of Helicobasidium mompa Isolates 177

10 crn

20 crn 1.5 crn < >t--)

plastic bag acrylic plate

I. .I -

carrdt inocbium VerAicuiite

Front Side Fig. 2. Schematic drawing of root box method using

carrots.

Two transparent acrylic plates (19.7 cm wide, 10 cm long) were put into a plastic bag (0.1 mm thick, 215 mm wide, 10 cm high) to make a root box (1.5 cm thick). Vermiculite or another medium filled the space between the two plates. Two carrots (cv. Kinko-Sanzun) raised in a greenhouse for 3-4 months were transplanted into the box. A piece of fungal inoculum growing on mulberry fragments was buried in contact with each carrot (Fig. 2). The bottom of the plastic bag was cut at each corner for drainage. Inoculated carrots were incubated in a green- house at 25°C. The surface of the carrots was observed weekly through the plate with the unaided eye or with a magnifying glass to rate disease development for 14 weeks. Carrots with hyphal growth on the surface were rated as DI 1 and those covered with mycelial mats (Fig. 1D) or with infection cushions, DI 2. A partial or overall rot of carrots without hyphal growth of H. mompa occa- sionally occurred (referred to as spontaneous rot). Car- rots with overall spontaneous rot were immediately removed and excluded from the disease rating. Those with DI 2 were removed to confirm the rating. The rest of the carrots were removed for microscopic observations at the end of the experiment (14 weeks after inoculation) regardless of disease development. Virulence of isolates was rated according to the length of time to attain DI 2 among 12 replicates for each isolate or by the mean DI.

RESULTS AND DISCUSSION

Conventional method Symptoms on plant tops Symptoms on plant tops

were of two types : most commonly, all leaves suddenly turned brown and hung down on branches or rarely, all leaves yellowed and subsequently fell. In the latter type, a few green leaves often remained on the tips of branches. Only four plants inoculated with four different isolates

Table 2. Number of apple stocks killed during the first 6 monthsa)

Total Weeksb) 0-13 14 16 17 18 19 20 21 23 24 25 26 Isolate

a) A total of 19 stocks were used for each isolate. b) Plants were inoculated in early April 1999.

had the yellowing symptoms. There was no relationship between the symptom types and isolates or between the types and the time required for symptom development. The browning and the yellowing types corresponded to the acute wilt and acute defoliation types, respectively, according to Fujita3) who classified disease symptoms observed in orchards on naturally infected apple trees with dwarf stock into four types.

Thirty-six of 456 apple stocks used for both trials were killed by violet root rot within 6 months after inoculation (Table 2). Infection cushions were found beneath the hyphal mat. Isolate 12 had killed six and seven plants, isolate 11 two and five plants by weeks 14 and 26, respectively. Isolates causing wilt early in the experi- ments tended to kill many plants by the end. Isolate 13 was an exception, killing two plants in the fall of the first year (25 and 26 weeks after inoculation).

All plants extended leaves till the end of trial 2 in early June (in the second year), although 20 plants inoculated with 15 different isolates did not resume growth till the beginning of May. Among these 20, 8 were rated as DI 2

178 JGPP

at the end of trial 2, whereas 36 other plants with normal flush were also rated as DI 2. Because substantial damage does not occur without the formation of infection cush- ions4,6, 12) , i. e., DI 2, the difference in the time of flush may not solely be ascribed to the damage of ~ d e ~ ~ o u n d plant parts, but the development of new roots may differ from plant to plant. These results suggest that the damage on underground parts does not always correlate to above- ground symptoms.

A mycelical mat covered the whole stem base and the basal part of most roots on wilted plants. Mycelial mats and infection cush- ions at the stem base were often degraded, and traces of infection cushions remained as holes in the bark (Fig. IB and C) , beneath which cortical tissues had disintegrated. These holes were already evident a t the end of trial 1 and became more prominent in trial 2. In the latter trial, the holes occurred on some parts of the bark, and new infec-

Signs on underground parts

100

80

- 60

2 C w 3 0;

U 2 40

20

0 Trial 1 Trial 2

tion cushions formed on other parts. The number of plants with no signs (DI 0) did not

increase in the 8 months from October (trial 1) to June (trial 2), even though disease severity of infected plant8

Fig. 3. Change in disease severity of apple plants esti- mated 6 months (trial 1) and 14 months (trial 2 ) after inoculation. DI 0, no sign ; DI 1, with hyphae andlor rnycelial strands ; DI 1.5, with mycelial mat ; DI 2 , with infection cushions ; DI 3, plants wilted.

Trial 1 (Apr 1999 - Oct 1999)

9 8

2 6 a 5 5 k 4

1 2

$ 7

n E 3

1 0

10 9 8

3 6 0 5

f 7

L

: 4 5 3

2 1 0

z

2.5

2

3.5 g

1 s

0.5

0

Isolate

Trial 2 (Apr 1999 - Jun 2000)

2.5

2

1.5 8 1 =

0.5

0

Fig. 4. Disease indices of apple plants in trials 1 and 2 €or each isolate. DI 0, no sign ; DI 1, with hyphae and/or mycelial strands ; DI 1.5, with mycelial mat ; DI 2, with infection cushions ; DI 3, plants wilted.

Estimating Virulence of Helicobasidium mompa Isolates 179

became slightly worse (Fig. 3). These facts indicate that the fungus remains inactive during plant dormancy.

0

2

4

6 r

i s 10

12

14

12 , I 1

c 2

1.6

- e

s 0.5

0 2 4 6 1 1 12 13 14 17 18 21 48 70 136 169 189

I80 I ate

Fig. 5 . Comparison of virulence on carrots estimated by three different criteria using the root box meth- od. A, shortest time to attain DI 2 ; B, disease indices of inoculated carrots for each isolate at the end of experiments (13 weeks after inocula- tion) ; C, mean DI at the end of experiments.

Observations from trial 1 suggest that the fungus is active in summer. With rising temperature in spring, the fungus is considered to become active, producing infection cush- ions. High temperatures in mid-summer promote desicca- tion and wilting of leaves. These facts are in accordance with observations on naturally infected trees on which symptoms and signs become evident in the summer3).

Disease indices of apple plants in trials 1 and 2 were illustrated for each isolate (Fig. 4). Isolates 10, 17, 3, 1, 26, 35, 136 and 14 did not form infection cushions during the experiments and were considered to be weakly virulent. Isolates 4, 6, 5, 48, 189, 70, 18 and 32 had an average DI of 0.9-1.4 ; their virulence was estimated as moderate. They caused wilt of one or two plants, i .e. , DI 3, and more than 60% of the plants lacked infection cushions. Isolates 13, 2, 38, 169, 11, 21 and 12 were regarded as highly virulent. These isolates killed (wilted) more than three plants, and infection cushions formed on more than one-third of the plants.

Disease severity varied among replicate plants inocu- lated with the same isolates. For example, inoculation with isolate 11 in trial 1 resulted in the death (DI 3) of three plants, infection cushion formation (DI 2) on four plants, hyphal growth (DI 1) on one plant, and no hyphal growth on one plant (DI 0). The results were consistent in trial 2.

Root box method Soil

Virulence

Three kinds of media, i .e. , vermiculite, Ka- numa soil and perlite, were tested using nine isolates to determine which medium provided the most stable results in the root box method. Infection cushions devel- oped on 22.2, 78.1 and 62.5% of carrots in Kanuma soil, vermiculite and perlite, respectively. Fungal growth was best and easy to observe in perlite, which was difficult to handle for the root box method; perlite was easily knocked out when the box was disturbed for observation. Consequently, vermiculite was selected for the root box medium.

Fifteen isolates were Determination of virulence

Table 3. Effect of unsterile field soil on spontaneous rot, vigor of carrots and growth of miscellaneous fungi on the inoculuma)

Medium Vigor of carrotsC)

Vermiculite only 7 0.3 1.7 Vermiculite + unsterile soil 2 1.0 0.1

No. carrots with spontaneous rotb) fungus on inoculumd)

Growth of miscellaneous

a) Ten % (v/v) of unsterile field soil was added to vermiculite in the root box method. A total of 24 carrots in 12 root boxes were used for each treatment. Isolate 18 was used.

b) Determined 5 weeks after inoculation. c) Mean number of carrots with fully developed leaves determined 2 weeks after inoculation. d) Growth of miscellaneous fungi on mulberry twig inoculum was rated as follows ; 0 : no fungal growth, 1 : fungal growth

on one end of the inoculum, 2 : fungal growth on both ends, and 3 : fungal growth over the inoculum. Determined 3 weeks after inoculation.

180 JGPP

Isolateb) 10 26 13 17 35 48

Table 4. Comparison of virulence level estimated by conqentional method with apple stocks and root box method with carrots

70 12 2 4 5 14 38 169 6 21 18

189 11 136

Conventional method Virulence level Low Moderate High I I

I I

Mean DI") 0.9 I 11.5 2.2 Isolateb) 10 17 1 3 26 35 136 141 6 4 5 48 32 18 189 701169 13 2 38 21 11 12 No. dead trees 0 0 0 0 0 0 0 0 1 1 1 1 0 2 2 1 1 1 4 2 5 2 2 5 7

Root box method Virulence level Low High I Week") 11 10 9 1 8 7 6 5 4 3

used, and the virulence was estimated by three criteria for each isolate (Fig. 5A-C) : A) shortest time to attain DI 2 among 12 replications (Fig. 5A) ; B) disease indices of inoculated carrots for each isolate at the end of experi- ments (13 weeks after inoculation) (Fig. 5B) ; and C) the mean DI at the end of experiments (Fig. 5C). The de- crease in the number of replications because of spontane- ous rot biased the estimate based on criterion C. For example, on the basis of criterion C, isolates 13 and 48 were over-estimated since the number of carrots was not enough to present the data as mean DI. On the other hand, isolate 12 was under-estimated because it produced infection cushions 7 weeks after inoculation. Consequent- ly, the level of virulence was estimated using the root box method and the time DI 2 was first recorded among replications.

Unsterile field soil (lo%, v/v) was added to vermiculite to alleviate sponta- neous rot. Addition of unsterile soil reduced spontaneous rot as well as the growth of miscellaneous fungi on the inoculum of isolate 18 and improved the vigor of carrots (Table 3). Only two of 24 carrots had spontaneous rot in soil-amended vermiculite, whereas seven rotted sponta- neously in vermiculite. Unsterile soil also improved the vigor of carrots as determined by the number of fully developed leaves 2 weeks after inoculation and sup- pressed the growth of miscellaneous fungi on the inocu- lum. These fungi colonized 22 inoculum fragments in vermiculite and three in soil-amended vermiculite. Since the production of infection cushions (DI 2) by isolate 18 first occurred 6 weeks after inoculation in both treat- ments, the estimate of the isolate was not affected by the

Minimizing spontaneous rot

presence or absence of unsterile soil. Comparison between the conventional method and

the root box method Table 4 summarizes the viru- lence of each isolate estimated by the conventional method and by the root box method. According to the results from the conventional method, isolates were divided into three categories, i.e., virulence low : no apple plant killed, mean DI < 0.9 ; virulence moderate : 0-2 plants killed, mean DI < 1.5 ; and virulence high : more than two plants killed, mean DIL1.5. The shortest time to attain DI 2 among replications by the root box method is illustrated for each isolate based on the results from six trials with 7 to 15 isolates in different combinations for each trial. Isolates 14 and 18 were most virulent, attain- ing DI 2, 3 weeks after inoculation. Carrots inoculated with isolates 10, 13, 17, 35 and 48 did not have infection or mycelial mats (DI 2) within the experimental period (10 to 14 weeks after inoculation). Most isolates catego- rized as highly or moderately virulent by the conventional method caused DI 2 within 8 weeks after inoculation by the root box method. Most isolates estimated as weakly virulent in the former did not incite DI 2 within the experimental period for 10-14 weeks, with the exception of isolate 26, with DI 2, 11 weeks after inoculation. Consequently, isolates producing infection cushions with- in 8 weeks on at least one carrot out of 12 replicates may be regarded as highly virulent, and those producing infec- tion cushions in 9 weeks or later as weakly virulent.

Although the virulence levels determined by both methods were almost parallel, isolates 14 and 136 were estimated as highly virulent by the root box method, but as weakly virulent by the conventional method with apple

Estimating Virulence of Helicobasidium mompa Isolates 181

plants (Table 4). Isolates 13 and 48 were the reverse : weakly virulent on carrots and moderately to highly virulent on apple plants (Table 4). Because the root box method was developed to screen weakly virulent isolates with dsRNA to exploit them for biological control of apple tree violet root rot, the method is effective for primary screening of many isolates. The ultimate screeening should, however, be made on apple trees.

Isolates 14 and 136 originated from sweet potato and isolates 13 and 48 from apple tree (Table 1). The violet root rot fungus has been found on a wide range of hosts, including both woody and herbaceous plants7) and exists in the forest (H. Nakamura, unpublished). These facts suggest H. mompa is native to the forest, which repre- sents the most stable habitat with little disturbance. Sweet potato fields are highly disturbed with annual tillage ; strains that can adapt to frequent disturbance of the habitat may thus be selected. Differentiation in eco- logical strategy should be further examined in H. mompa. If there is a difference in strategy, then, the estimate by the root box method with carrots should be conducted carefully when isolates from annual herbaceous plants are used to infer virulence on woody plants.

CONCLUSION

Inoculation experiments using the conventional method on apple stocks revealed isolate variability in virulence. This variability was reproducible by the root box method with carrots. The difficulty in estimating virulence of the violet root rot fungus can be ascribed to the large variance among replicates in both methods. The lag phase before the fungus invaded host tissues was considered to be inevitable even when the fungus was placed in contact with the carrots. Suzui“) observed the infection process of this fungus on asparagus roots using root boxes and noted that in some cases, the roots were not colonized even though the fungus was in contact with roots for a month. H. mompa is fastidious, and various environmen- tal factors must be fulfilled to complete pathogenesis. Satisfying all the parameters during the long-term inocu- lation experiments with perennial plants is difficult ; not all the replicates had the same disease severity using the conventional method. The estimate by mean DI, however, did not always indicate the level of virulence using the root box method. Using time as the virulence parameter reduced the variation within replicates so that the poten- tial level of virulence for each isolate could be estimated. The root box method is also advantageous over the conventional method with apple plants in reducing time, space, and cost, as well as in allowing continuous observa- tions of disease progress. We did not examine varietal differences for carrot since modern carrot cultivars are all

susceptible to the related fungus H. purpureum2). How- ever, varietal differences should be examined to minimize the seasonal chhnge in growth rate.

ACKNOWLEDGMENTS This work was conducted by the program for Promotion of

Basic Research Activities for Innovative Biosciences.

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