TECHNICAL BACKGROUND

&null;0001&null; The present invention relates to the use of certain fungi in the enhancement of wood quality. More particularly, but not exclusively, it relates to the use of particular fungi strains of Ophiosotoma floccosum, Ophiostoma piceae or Ophiostoma pluruanulatum, or mixtures of strains, to elicit a useful effect on wood or wood products derived therefrom in or on a substrate or locum e.g. including biocontrol; the prevention of staining of cellulosic materials from detrimental strains or other detrimental effects from detrimental strains, or for the reduction of pitch components and/or their detrimental effects, the production of positive effects achieved from the strains in regard to solid wood with uptake of liquid materials including but not limited to glue, preservative, varnish, paint, improvement in mechanical or chemical pulping as achieved by increased efficiency of cooking liquor (with concomitant lower kappa number value achieved for the equivalent cooking liquor or less cooking liquor required for the same kappa number achieved without use of strains); and increased steam penetration efficiency. The kappa value is an indication of the amount of bleaching chemical required to remove the residual lignin.

BACKGROUND ART

&null;0002&null; Wood is a complex material composed of cellulose, hemicellulose, lignin, and wood extractives or a resinous material commonly called pitch. &null;Resin&null; or &null;pitch&null; (used interchangeably) includes that complex mixture of hydrophobic substances in wood, which are soluble in neutral organic solvents such as methylene chloride, diethyl ether, benzyl alcohol and the like. These include the terpenes, the diterpene (&null;resin&null;) acids, fatty acids and esters, glycerides and waxes as well as alcohols, hydrocarbons and other compounds associated therewith.

&null;0003&null; In the production of products from wood pulps, the presence of pitch is undesirable as it frequently forms deposits which are difficult to remove or its presence causes detrimental properties to the products derived from the wood.

&null;0004&null; Another problem in the timber industry derives from staining. When trees are cut down they commonly become infected by any one or more of a variety of fungi which can stain the wood in any one or more of a variety of colours. A major problem in the industry today involves loss of value in timber products, and any products derived therefrom due to the unsightly staining caused by so-called blue stain fungi which can colour the wood a variety of colours including gray, dark blue and black, such staining appearing in the wood even though the outer surfaces or regions of the wood have been cut away in forming the lumber.

&null;0005&null; In the early 1990's workers at Sandoz Chemicals Biotech Research Corporation and their colleagues at the University of Minnesota developed a resin degradative and biocontrol fungus for pretreatment of wood from an Ophiostoma species. This work resulted in the development of the current commercial product, the fungal inocula Catapip&null;97. It was made by classical mating of isolated ascospores from various Ophiostoma piliferum isolates of the United States. Ophiostoma piliferum is a saprophytic Ascomycete found throughout the world, and commonly referred to as one of the sapstain fungi.

&null;0006&null; Sapstain fungi grow mainly in wood in the ray parenchyma cells, within resin canals, within tracheids and fibre cells and penetrate simple and bordered pits, occasionally forming bore holes through wood cell walls. Sapstain fungi are not capable of degrading cellulose or lignin, but metabolise resin extractives, starch and simple sugars. Sapstain fungi cause a characteristic stain of sapwood resulting in a blue, black, grey or brown discolouration of the wood, and is responsible for major economic losses in the timber and some pulping industries. Problems with sapstain are most prevalent in warm, humid climates and when wood with high sapwood content is used (Kay et al, 1997).

&null;0007&null; Ascospore mating and selection procedures on loblolly pine produced the isolate Cartapip&null;97 which grew rapidly on wood chips, degraded substantial quantities of pitch and did not stain wood (Farrell et al, 1992; Wendler et al, 1992; Hoffmann et al, 1992; Farrell et al, 1993). This colourless (albino) isolate was shown to be blocked in the 1,8-dihydroxynaphthalene (DHN) melanin synthetic pathway by the inability to produce the intermediate scytalone (Zimmerman et al 1993). Later work showed that O. piceae albino strains could also be generated by the same methodology (White-McDougall 1998). Treatments of wood chips with the albino fungi resulted in a biocontrol effect, such that the albino fungus quick grew throughout the chips and suppressed the growth of staining fungi (Farrell et al, 1993). Biocontrol of sapstain in logs using the Cartapip 97 inoculum was demonstrated on red pine in the lab and in th field (Benhrendt et al 1995; Behrendt et al, 1995).

DISCLOSURE OF THE INVENTION

&null;0008&null; It is an object of the present invention to provide an inoculum and a process which enhances wood quality, having advantages over those previously known, to provide a process which enhances wood quality of wood (including but not limited to New Zealand woods or woods of the Souther Hemisphere), or to at least provide the public with a useful alternative.

&null;0009&null; Our recent studies dealing with New Zealand sapstaining organisms have yielded surprising results. Studies particularly with radiata pine have shown certain New Zealand Ophiostoma albino species especially albino or very slightly pigmented in their hyphae; even if slightly pigmented in other fungal bodies provide improved results in biocontrol of sapstain and reduction of pitch. These species have shown to provide an improved result over that of Cartapip&null;97 on radiata pine. Cartapip&null;97 has proved to be somewhat but not fully effective to reduce stain to the desired industry target of <10% stain in 3 months. Furthermore, our studies have shown mixtures of more than one albino fungus have also been effective in increasing brightness of wood, and/or brightness increase in chemical pulp resulting from albino treatment of wood, and/or improved pulping efficiency such that lower kappa numbers were achieved with treatment of the wood with he albino strains.

&null;0010&null; As used hereinafter reference to specific stains means the following:

&null;0011&null; Ophiosotoma floccosum strain F13&null;that strain deposited as AGAL accession number NM00/12246 on 16/08/00

&null;0012&null; Ophiosotoma floccosum strain F40&null;that strain deposited as AGAL accession number NM00/12247 on 16/08/00

&null;0013&null; Ophiosotoma floccosum strain F71&null;that strain deposited as AGAL accession number NM00/12490 on 16/08/00

&null;0014&null; Ophiosotoma floccosum strain F80&null;that strain deposited as AGAL accession number NM00/12491 on 16/08/00

&null;0015&null; Ophiasotoma floccosum strain F93&null;that strain deposited as AGAL accession number NM00/12492 on 16/08/00

&null;0016&null; Ophiosotoma floccosum strain F3410&null;that strain deposited as AGAL accession number NM00/12252 on 16/08/00

&null;0017&null; Ophiosotoma floccosum strain F5040&null;that strain deposited as AGAL accession number NM00/12251 on 16/08/00

&null;0018&null; Ophiosotoma floccosum strain F4650&null;that strain deposited as AGAL accession number NM00/12253 on 16/08/00

&null;0019&null; Ophiosotoma floccosum strain F7073&null;that strain deposited as AGAL accession number NM00/12493 on 16/08/00

&null;0020&null; Ophiostoma piceae strain OPC 422&null;that strain deposited as AGAL accession number NM00/12248 on 16/08/00

&null;0021&null; Ophiostoma piceae strain OPC 580&null;that strain deposited as AGAL accession number NM00/12249 on 16/08/00

&null;0022&null; Ophiostoma piceae strain OPC 194&null;that strain deposited as AGAL accession number NM00/12250 on 16/08/00

&null;0023&null; Ophiostoma piceae strain OPC 703&null;that strain deposited as AGAL accession number NM00/12489 on 16/08/00

&null;0024&null; In a first aspect of the present invention there is provided an inoculum for wood comprising or including one or more (preferably biologically pure) forms of a fungal culture of the species Ophiostoma floccosum and/or Ophiostoma piceae and/or Ophiosotoma pluruanulatum effective to reduce the amount of colour staining caused by wood staining fungi.

&null;0025&null; Preferably said fungal culture is selected from the following strains:

&null;0026&null; Ophiosotoma floccosum F71,

&null;0027&null; Ophiosotoma floccosum F80,

&null;0028&null; Ophiosotoma floccosum F93,

&null;0029&null; Ophiosotoma floccosum F40,

&null;0030&null; Ophiosotoma floccosum F13,

&null;0031&null; Ophiostoma piceae OPC 703,

&null;0032&null; Ophiostoma piceae OPC 422,

&null;0033&null; Ophiostoma piceae OPC 580,

&null;0034&null; Ophiostoma piceae OPC 194,

&null;0035&null; Ophiostoma pluruanulatum 3410,

&null;0036&null; Ophiostoma pluruanulatum 7073,

&null;0037&null; Ophiostoma pluruanulatum 5040,

&null;0038&null; Ophiostoma pluruanulatum 4650

&null;0039&null; In one embodiment the wood source may be wood chips and the fungus or fungi may be applied by spraying the wood chips. In an alternative embodiment the wood source may be logs to be cut into structural wood, and inoculation includes inoculating at least one end of the logs, and preferably all around the logs' surfaces. In a further alternative embodiment the wood source may be structural wood and inoculation includes inoculating at least 60% of the surface area of the structural wood. In another alternative embodiment the wood source may be structural wood and inoculation includes inoculating at least 60% of the surface area of the Structural wood, which may then be later made into chips or a subsequent fibre product.

&null;0040&null; Preferably the wood to be inoculated may be a conifer such as but not limited to Radiata pine, Douglas fir or Loblolly pine, and it may also be a hardwood including but not limited to eucalyptus, oak, poplar, or aspen.

&null;0041&null; In a second aspect of the invention the is provided a biologically pure culture of a strain of Ophiostoma floccosum having all of the identifying characteristics of one of the fungi of strains F40, F13, F71, F80 and F93.

&null;0042&null; In a third aspect of the invention there is provided a biological pure culture of a strain of Ophiostoma piceae having all of the identifying characteristics of one of the fungi of strains OPC 703, OPC 422, OPC 580 and OPC 194.

&null;0043&null; In a fourth aspect of the invention there is provided a biologically pure culture of a strain of Ophiostoma pluruanulatum having all of the identifying characteristics of one of the fungi of strains 3410, 7073, 5040 and 4650.

&null;0044&null; In a fifth aspect of the invention there is provided a method of reducing the amount of colour staining caused by wood staining fungi in and/or on a wood source which comprises or includes inoculating at least a portion of the wood source with an effective amount of at least one fungus selected from the group consisting of the species Ophiosotoma floccosum, Ophiosotoma piceae and Ophiostoma pluruanulatum, which is capable of reducing the amount of colour staining caused by the wood staining fungi.

&null;0045&null; Preferably said fungi may be selected from the following stains:

&null;0046&null; Ophiosotoma floccosum F13,

&null;0047&null; Ophiosotoma floccosum F40,

&null;0048&null; Ophiosotoma floccosum F71,

&null;0049&null; Ophiosotoma floccosum F80,

&null;0050&null; Ophiosotoma floccosum F93,

&null;0051&null; Ophiostoma piceae OPC 703,

&null;0052&null; Ophiostoma piceae OPC 422,

&null;0053&null; Ophiostoma piceae OPC 580,

&null;0054&null; Ophiostoma piceae OPC 194,

&null;0055&null; Ophiostoma pluruanulatum 3410,

&null;0056&null; Ophiostoma pluruanulatum 7073,

&null;0057&null; Ophiostoma pluruanulatum 5040,

&null;0058&null; Ophiostoma pluruanulatum 4650

&null;0059&null; In one embodiment the wood source may be wood chips and the fungus or fungi may be applied by spraying the wood chips. In an alternative embodiment the wood source may be logs to be cut into structural wood, and inoculation includes inoculating at least one end of the logs, and preferably all around the logs' surfaces. In a further alternative embodiment the wood source may be structural wood and inoculation includes inoculating at least 60% of the surface area of the structural wood.

&null;0060&null; Preferably the wood source may derive from a conifer such as but not limited to, Radiata pine, Douglas fir or Loblolly pine and it may also be a hardwood including but not limited to eucalyptus, oak, poplar, or aspen.

&null;0061&null; In a sixth aspect of the present invention there is provided an inoculum for wood comprising or including one or more (preferably biologically pure) forms of a fungal culture of the species Ophiostoma floccosum, and/or Ophiostoma piceae and/or Ophiosotoma pluruanulatum effective in reducing the pitch content of wood.

&null;0062&null; Preferably said fling may be selected from the following strains:

&null;0063&null; Ophiosotoma floccosum F40

&null;0064&null; Ophiosotoma floccosum F13

&null;0065&null; Ophiostoma piceae OPC 422

&null;0066&null; Ophiostoma piceae OPC 580

&null;0067&null; Ophiostoma piceae OPC 194

&null;0068&null; In one embodiment of the wood source may be wood chips and the fungus or fungi may be applied by spraying the wood chips. In an alternative embodiment the wood source may be logs to be cut into structural wood, and inoculation includes inoculating at least one end of the logs. In a further alternative embodiment the wood source may be structural wood and inoculation includes inoculating at least 60% of the surface area of the structural wood.

&null;0069&null; Preferably the wood to be inoculated may be but is not limited to a conifer such as Radiata pine, Douglas fir or Loblolly pine and it may also be a hardwood including but not limited to eucalyptus, oak, poplar, or aspen.

&null;0070&null; In a seventh aspect of the invention there is provided a biologically pure culture of a strain of Ophiostoma floccosum having all of the identifying characteristics of one of the fungi of strains F40 and F13.

&null;0071&null; In an eighth aspect of the invention there is provided a biologically pure culture of a strain of Ophiostoma piceae having all of the identifying characteristics of one of the fungi of strains OPC 422, OPC 580, OPC 194.

&null;0072&null; In a ninth aspect of the invention there is provided a method for reducing the pitch content in a wood source and/or improving brightness in chemical pulp resulting from the albino treatment of wood, and/or improving pulping efficiency such that lower kappa numbers were achieved with treatment of the wood with the albino strains, which comprises or includes inoculating at least a portion of the wood source with an effective amount of at least one fungus selected from the group consisting of the species Ophiosotoma floccosum, Ophiosotoma piceae and Ophiostoma pluruanulatum, which is capable of reducing the pitch content.

&null;0073&null; Preferably said fungi may be selected from the following strains.

&null;0074&null; Ophiosotoma floccosum F40

&null;0075&null; Ophiosotoma floccosum F13

&null;0076&null; Ophiostoma piceae OPC 422

&null;0077&null; Ophiostoma piceae OPC 580

&null;0078&null; Ophiostoma piceae OPC 194

&null;0079&null; Preferably the method may further include the step of maintaining the inoculated wood source under conditions which allow fungal growth from the inoculation for a term sufficient to effect a reduction of the pitch content of the wood source by such inoculated fungal growth.

&null;0080&null; In one embodiment the wood source may be pulpwood, or unsterilised pulpwood, or unsterilised refined pulpwood. Alternatively the wood source may be wood chips and the fungus or fungi is applied by spraying the wood chips. Alternatively the wood source may be debarked or undebarked timber or logs.

&null;0081&null; Preferably the wood source may derive from but not limited to a conifer such as Radiata pine, Douglas fir or Loblolly pine and it may also be a hardwood including but not limited to eucalyptus, oak, poplar, or aspen.

&null;0082&null; Preferably the method may include using or applying more than one fungal strain.

&null;0083&null; In a tenth aspect of the invention there is provided a biological pure culture of a strain of Ophiostoma floccosum having all of the identifying characteristics of one of the fungi of AGAL Accession Numbers NM00/12246, NM00/12247, NM00/12490, NM00/112491 or NM00/12492.

&null;0084&null; In an eleventh aspect of the invention there is provided a biologically pure culture of a strain of Ophiostoma piceae having all of the identifying characteristics of one of the fungi of AGAL Accession Numbers NM00/12248, NM00/12249, NM00/12250 or NM00/12489.

&null;0085&null; In a twelfth aspect of the invention there is provided a biologically pure culture of a strain of Ophiostoma pluruanulatum having all of the identifying characteristics of one of the fungi of AGAL Accession Numbers NM00/1225, NM00/12252, NM00/12253 or NM00/12493.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE

&null;0086&null; From the 21 sapstain fungal species isolated and identified in New Zealand (Farrell et al, 1998), four species were chosen for a breeding programme in order to make albinos/white fungi. These New Zealand albinos were made as had been done with Cartapip 97, classically mating a variety of isolates of the same species to produce albino, non-melanising strains. Neither genetic engineering nor mutagens were used to make the isolates. The albino isolates were studied for their ability to biocontrol and reduce staining in mature radiata pine logs (Farrell et al 1997a; Farrell et at 1997b), and for their ability to reduce resin components in radiata pine wood chips.

&null;0087&null; Surprisingly and a significant advancement over what is presently commercially available, were the effect of some of the Ophiostoma floccosum, O. pluruanulatum, and O. Piceae albinos. In this example these stains were isolated from New Zealand but it is expected that these species and their subsequent albinos could prove beneficial when isolated from any country and origin and give beneficial effects superior to Cartapip&null;97, Some of the New Zealand albinos were significantly better at maintaining biocontrol effect and exclusion of detrimental organisms, including stain fungi, and degrading detrimental wood components than others and better than the commercial product. Also surprising and unobvious according to presently granted patents was the fact that different albinos of various species had differing degrees of effect, with some of the albinos providing other than a positive effect on wood with their application and incubation.

FIGURES

&null;0088&null; FIG. 1: Results of solid logs with albino Inoculation.

&null;0089&null; FIG. 2: 80,000 ton trial&null;Extractives of chips as a function of incubation time in the chip piles.

&null;0090&null; FIG. 3: Chips inoculated with both OF40 and OF13.

Example 1

&null;0091&null; Competition Assays

&null;0092&null; Albinos were constructed by single ascospore isolation and mating, according to Zimmerman, W. C., Blanchette, R. A., Burnes, T. A., Farrell, R. L. (1993). Melanin and perithecial development in Ophiostoma piliferum. Mycologia 87: 857-863.

&null;0093&null; Albinos were tested in the laboratory for presence of pigment under a variety of incubating conditions and in competition assays. The competition assays were conducted by inoculating either sterilised, gamma irradiated or some other method, or non-sterilised radiata pine cubes or chips with each or more than one of sapstaining species, including but not limited to Sphaeroides sapinea, Ophiostoma piliferum or Leptographium species, prior to, simultaneously or after inoculation with the albino or more than one albino fungus. In most assays the staining fungus/fungi was introduced to the chips as actively growing vegetative cells at one-tenth the cell density as the albino(s), an extreme situation with respect to challenge.

&null;0094&null; An example below shows typical conducted lab competition assays, which consisted of albinos made from New Zealand ascomycetes, and the commercial product Cartapip&null;897.

1

TABLE 1

Intensity

Comments

F71

0

F71 &null; Stainer same day

0.8

successful

F71 &null; Stainer next day

0.5

successful

F73

0

F73 &null; Stainer same day

1

successful

F73 &null; Stainer next day

1.3

Not successful

F74

0.5

Not successful

F74 &null; Stainer same day

1.5

Not successful

F74 &null; Stainer next day

1.5

Not successful

F77

0

F77 &null; Stainer same day

2

Not successful

F77 &null; Stainer next day

0.5

successful

F80

0.5

Successful

F80 &null; Stainer same day

1

Successful

F80 &null; Stainer next day

1

Successful

F81

0

Successful

F81 &null; Stainer same day

2

Not Successful

F81 &null; Stainer next day

2

Not Successful

F87

0

Successful

F87 &null; Stainer same day

1.2

Not successful

F87 &null; Stainer next day

1.5

Not successful

F93

0

Successful

F93 &null; Stainer same day

0.3

successful

F93 &null; Stainer next day

0.5

successful

OPC703

0.2

Not successful

OPC703 &null; Stainer same day

0.3

successful

OPC703 &null; Stainer next day

0.7

successful

OPC422

0.5

Not successful

OPC422 &null; Stainer same day

0.8

successful

OPC422 &null; Stainer next day

1

successful

Intensity

% coverage

OPC194

0

successful

OPC194 &null; Stainer same day

3

Not successful

OPC194 &null; Stainer next day

3

Not successful

OPC580

0

successful

OPC580 &null; Stainer same day

2

Not successful

OPC580 &null; Stainer next day

1.8

Not successful

3410

0

Successful

3410 &null; Stainer same day

0.2

Successful

Intensity

Comments

3410 &null; Stainer next day

0.5

Successful

7073

0

Successful

7073 &null; Stainer same day

0.5

Successful

7073 &null; Stainer next day

0.7

Successful

Cartapip 97

0.2

Not successful

Cartapip &null; Stainer same day

0.7

successful

Cartapip &null; Stainer next day

1.7

Not successful

Stainer same day

3.0

Stainer next day

3.3

Uninoculated sterile control

0

&null;0095&null; Albino concentration was 106 blastospore/ml

&null;0096&null; Stainers (fungi which cause stain including but not limited to Leptographium, or Diplodia (Sphaeropsis sapinea) or any other organism that leaves a sin on wood) concentration was 106 blastospore/ml

Example 1

&null;0097&null; Results

&null;0098&null; Surprisingly not all of the albinos had the same effect for prevention of stain. This was an unexpected and unobvious result as previously it was considered that any albino or white fungus, or almost albino would constitute a biocontrol effect on wood, and prevent staining fungi from staining the wood, Successful competitive albino fungi were judged as those with less than 0.5 stain score on their own, and that maintained a stain score when challenged with one of the staining fungi, in this example Leptographium or Diplodia (Sphaeropsis sapinea) of 1 or less than 1. As shown in the results, the preferred organisms were F71, F80, F93 (all albinos, with regard to hyphae but not with regard to synnemata, of Ophiosotoma floccosum), OPC 703, 422 (albinos, with regard to hyphae but not with regard to synnemata, of the species Ophiostoma piceae) and 3410 and 7073 (albinos, with regard to hyphae but not with regard to synnemata, of the species Ophiostoma pluruanulatum). The commercial Product Cartapip&null;97. had too much stain and was not successful.

Example 2

&null;0099&null; In order to teach the mechanism of positive effect upon wood, the albino fungi have been tested in the laboratory for growth characteristics and enzyme production, By standard biochemistry assays the albino fungi have been tested for their ability to deplete starch in growth media, and units of activity in media of amylase and lipase enzymes.

2

TABLE 2

Albino Characteristic Chart

Key Characteristics of New Zealand Ophiostoma albinos:

Nutrient depletion

Cube Competition

Albino

% Starch

Amylase

Liplo

O.pilaf

Growth in

Species

ID &null;

Depletion/hr

Lipase

Lepto(L)

(D)

(Op)

Blocks

pluruanulanim

3410

92/24

700

342

3440

98/24

364

5040

2226

486

4630

1620

4650

540

4680

1119

673

4880

738

4890

88/24

413

5980

97/24

576

7052

100/24

840

643

1

1

1

7058

92/24

917

701

1

1

1

7060

85/100

843

566

1

1

1

7073

721

1

1

1

7076

98/48

1527

384

1

1

1

7078

1041

2

1

floccosum

F3

74

1

1

P

F13

293

1

1

P

F29

270

496

2

1

P

F36

44/24

226

653

1

1

P

F61

92/24

230

526

P

F63

21/24

235

365

P

F30

45/24

388

482

1

1

P

F20

16/24

502

562

1

1

1

P

F35

100/48

959

604

1

1

P

F40

17/24

462

728

1

1

1

P

piccae

1

156

piccae

194

14/24

180

442

P

422

25/24

177

421

1

P

542

13/24

1091

782

1

1

P

400

95/24

1055

702

P

12

553

1

P

580

11/24

960

614

2

P

&null;0100&null; Stain during competition is marked on a scale of 1-5 with 0 being white wood and 5 black stain. Each competitive experiment is marked individually.

Example 2

&null;0101&null; Results

&null;0102&null; There is a variety of responses of the albino fungi to these diagnostic tests. Not all the albinos have the same characteristics.

Example 3

&null;0103&null; Field Results on Solid Logs with Albino Inoculation

&null;0104&null; Albino fungi were grown in liquid cultures of growth media consisting of sufficient carbon, nitrogen and trace elements and vitamins to promote cell biomass accumulation (standard and non-standard mycological media are acceptable), either used in the spent growth media, or harvested by centrifugation and resuspended in water or buffer, with or without washing, were sprayed onto radiata pine logs, Either a pure single strain or more than one strain can be used simultaneously or consecutively. The typical cell concentration of the fungus in spent media, water or buffer was from 10e2 to 10e6 colony forming units per millilitre. The fungal cell suspensions were applied onto the logs by back-pack spray systems, or commercial sprayers, either variety such as used by commercial agricultural spray systems or commercial log spray systems. Fungal cell suspensions could also be applied to logs by dipping the logs into a bath suspension containing the cells in water, buffer or spent growth media. Fungal application can be done only one time or more than one time. Mouldicides may be used after a few days of the fungal application to improve surface appearance. After application of the fungal cell suspension to the logs, the logs were let in the field, or in an enclosed container such as warehouse, boat etc, and after 1, 2, 3 or longer months the amount of in was assessed on the logs visually. Results of one such application of multiple fungi are given in FIG. 1.

Example 3

&null;0105&null; Results

&null;0106&null; Some albinos can have a positive effect on wood quality, biocontrol and keep wood from staining, better than the commercial fungus and better than commercial anti-sapstain chemicals. Some albinos have detrimental effects and increase the amount of stain, therefore not all white fungi behave in the same manner with regard to their effect on wood.

EXAMPLE 4

&null;0107&null; Resin Component Degradation

&null;0108&null; Solid wood or wood chips, either previously sterilised by gamma irradiation or some other method, or non-sterilised, was treated with more than two dozen albino fungi, a bacteria (for example Pseudomonas resinovorans) or the commercial product Cartapip 97. The inoculated wood was either left to stand at ambient temperature or incubated from 0 degrees Centigrade to plus 65 degrees Centigrade. The wood was analysed anytime from 2 days to 16 weeks, most typically between 1-8 weeks) for full extractive analysis.

&null;0109&null; Extractives Analysis

&null;0110&null; The wood extractives content of treated and untreated chips was determined on freeze dried chip samples which had been ground to <0.5 mm. The wood meal was extracted by solvent extraction with acetone using a Soxtec extractor. Gas chromatography of the methylated extract was performed using a DB-1 5 m&null;0.32 mm capillary column fitted with a 0.5 m&null;0.5 mm deactivated silica retention gap and using an on column injection technique. Detection was by flame ionisation detector and quantitation was performed using the internal standard technique on the chromatography data system.

Example 4

&null;0111&null; Results

&null;0112&null; Control, non-fugal inoculated radiata pine wood chips were analysed as a reference for resin acids, fatty acids and glycerides, and the results are given in Table 3,

3

TABLE 3

Analysis of Wood Extractives of Control Chips from Lab Experiment

Sample

% Acetone

% Free

% Resin

%

Control

1.26

0.227

0.411

0.130

Control

1.27

0.156

0.282

0.081

Average:

1.26

0.19

0.35

0.11

&null;0113&null; Results for the fungal treated samples can be most easily compared as to the effect of one fungal isolate as compared to another fungal, rather than compared to the reference control, non-fungal treated samples.

&null;0114&null; Dozens of albino isolates were treated for their ability in the laboratory to degrade wood extractives. Results given in Table 2 show in duplicate some of the better and the lesser fungal overall effects for wood extractives decrease.

4

TABLE 4

Analysis of Wood Extractives after Fungal Treatment

%

% Free

Acetone

Fatty

% Resin

Sample

Extractives

Acids

Acids

% Glycerides

OPC 542

1.46

0.092

0.730

0.025

&null;

1.70

0.096

0.797

0.018

F63

1.19

0.108

0.481

0.020

&null;

1.02

0.080

0.375

0.021

7060

0.73

0.039

0.226

0.012

&null;

0.74

0.040

0.222

0.015

7073

0.72

0.073

0.184

0.039

&null;

0.68

0.069

0.168

0.036

7076

1.19

0.120

0.268

0.093

&null;

1.15

0.120

0.265

0.089

OPC 422

0.57

0.044

0.133

0.033

&null;

0.61

0.048

0.150

0.038

F13

0.62

0.036

0.100

0.016

&null;

0.64

0.036

0.095

0.017

OPC 580

0.65

0.038

0.165

0.010

&null;

0.70

0.050

0.217

0.015

F40

0.37

0.028

0.129

0.017

&null;

0.58

0.043

0.156

0.023

&null;0115&null; As shown in Table 4, the various isolates tested on wood chips varied considerably in their ability to degrade wood extractives. The range of extractives values for differing fungal treatments was from 0.37% to 1.70% by weight of wood; obviously some much more efficient at degrading the wood extractives than others. Fungi such as the isolate O. floccosum F40 on the average decreased total acetone extractives to 0.47% versus OPC 542, with the same length of time incubating on the chips, had an average acetone extractives value of 1.58% by weight of wood. Analogously, some of the fungi had significant decreases in free fatty acids, for example O. floccosum F40 with free fatty acids of 0.035% and free resin acids of 0.143% whereas O. floccosum isolate F63 did not decrease the free acids as much with average % free fatty acids of 0.094% and % free resin acids of 0.428% by weight of wood.

&null;0116&null; The free fatty acids and free resin acids were further analysed by gas chromatography. Examples of the data for isolate F13 are given in Table 5.

&null;0117&null; All the data obtained to date on wood extractive decrease was sorted for the possible contenders for further evaluation on the basis that their resin acid contents were below 0.2% by weight of wood; These isolates were O. floccosum F40 and F13, and O. piceae C OPC 422, OPC 580, and OPC 194.

5

TABLE 5

Analysis of Free acids and extractive components after F13 Lab Fungal

Treatment-duplicate analysis

% Extractives

0.62

0.64

mg Analysed

0.546

0.574

C16:0 found (ug)

4.76

4.96

C18:2 found (ug)

5.94

5.51

C18:1 found (ug)

19.88

20.53

C18:0 found (ug)

4.01

4.09

Free Fatty Acids Sum (ug)

34.59

35.09

% Free Fatty Acids in Extract

6.34

6.11

Corrected % FFA in Extract

5.82

5.60

% Free Fatty Acids in Sample

0.039

0.039

Corrected % FFA in Sample

0.036

0.036

Pimaric found (ug)

15.31

14.73

Sandara. found (ug)

3.19

3.13

Isopimaric found (ug)

8.34

7.59

Levo. &null; Pal. found (ug)

16.53

16.02

Dehydro. found (ug)

44.26

42.96

Abietic found (ug)

2.94

3.04

Neoabietic found (ug)

5.81

5.59

Resin Acids Total (ug)

96.38

93.06

% Resin Acids in Extract

17.65

16.21

Corrected % RA in Extract

16.20

14.85

% Resin Acids in Sample

0.109

0.104

Corrected % RA in Sample

0.100

0.095

C16:0 found (ug)

3 Unidentified (ug)

14.62

16.47

C18:1 found (ug)

C18:2 found (ug)

C18:0 found (ug)

Glycerides Sum (ug)

14.62

16.47

% Glycerides in Extract

2.68

2.87

Corrected % Glyc. in

2.52

2.68

% Glycerides in Sample

0.017

0.018

Corrected % Glyc. in

0.016

0.017

% Extract Identified

24.5

23.1

Example 5

&null;0118&null; Effect of Albinos on Processed Pulp

&null;0119&null; Fungal cell cultures as described in Example 1 were applied either to logs or to chips at a ratio of 1000 gallons cell suspension in water to 200 tons wet weight chips&null;this cell suspension to wood weight ratio could be varied by several orders of magnitude.

&null;0120&null; In this Example are given the results from the use of two O. floccosum albinos mixed together and applied onto chips, or three O. piceae albino fungi mixed together and sprayed onto wood chips. Control pile of chips sprayed just with water were also established.

&null;0121&null; OF Pile: in this example consisted of chips sprayed with water plus a mixture of two Ophiostoma albinos, O. floccosum 40, and O. floccosum 13, but other combination of albino fungi could also be used. OPC Pile in this example consisted of chips sprayed with water plus mixture of three O. piceae C albino isolates, OPC 580, OPC 422 and OPC 194 cell concentrate, but other combination of albino fungi could also be used.

&null;0122&null; Sprayed in 4500 litre water solution onto 200 tons wet weight chips. Temperature or rain/moisture does not affect the spray trials.

&null;0123&null; Materials and Methods

&null;0124&null; Survey, Culture Isolations and Identification, and Competition Experiments.

&null;0125&null; Sampling, isolations, identifications, mating, culture growth and competition experiments on wood cubes and chips were done as described by Farrell, et al, 1998

&null;0126&null; Analysis of the Mill Trial

&null;0127&null; Spray Inocula for the Mill trial

&null;0128&null; The O. piceae 540, O. piceae 422, O. piceae 194 culture suspensions were mixed together and applied to one pile, and O. floccosum 40 and O. floccosum 13 were mixed together and applied to another pile, as described in the Materials and Methods and details in Appendix 2. From the spray tank, a sample of the inocula was taken at start middle and end of the spraying of the 200-ton wet weight of chips, to verify the mixing and dosage of the blastospores per volume spray was consistent throughout the trial.

&null;0129&null; Fungal Growth during Eight Week Trial

&null;0130&null; Growth of the fungi at the mill piles was monitored by standard mycological culturing of chips taken from the four piles every two weeks during the trial and with many isolates checking with molecular markers for DNA analysis. Consistently, the albino O. floccosum grew from the OF pile. There was little if any growth from the OPC pile of O. piceae and there was O. floccosum, growing in this pile, the native O. floccosum of the site The Wet Pile showed positive Ophiostoma identification of native O. quercus, O. floccosum Rhizopus; Trichoderma, Fusarium and yeasts. Therefore the Tantanoola Mill site consistently has the presence of moulds, yeasts and three sapstain organisms O. quercus, O. floccosum and Graphium species C on the radiata pine wood chips.

&null;0131&null; Analysis of the Pulps of the Mill Trial

&null;0132&null; DNA: The wood chips of the piles were made into pulp at the mill by normal procedures. Samples of pulp were taken for further analysis from the Chemiwasher, Twin wire wash press (TWWP), and bleached pulp (M57). DNA extractions were performed on the pulps to show whether there was any trace of the New Zealand-origin fungi; these analyses showed that there was no residual DNA left in the pulps. Polymerase chain reaction (PCR) of specific DNA probes for O. piceae and O. foccosum on the extractions confirmed that no DNA could be isolated from the pulps, and hence no PCR reaction was observed; therefore there was no residual DNA in the pulps from the applied albino fungi.

&null;0133&null; EXTRACTIONS: Soxtec dichloromethane extractions of the pulps from the chips of the 8 week mill trial (Dry pile not processed) were compared to the mill data from May 1999 to February 2000, with seasoning of chips routinely during this period being 12-16 weeks and results are given in Table 6.

6

TABLE 6

Wood Extractives of Pulps from Mill

(average values based on wood weight)

SampleChemiwasherTWWPM57Mill data

May '99-Feb 2000

0.90%

0.10%

0.07%

OF Pile pulp

0.92

0.12%

0.05%

OPC Pile pulp

0.90%

0.12%

0.06%

Wet Pile pulp

0.88%

0.10%

not done

&null;0134&null; TWWP&null;Twin wire wash press

&null;0135&null; Interestingly, as evident in the data of Table 6, it can be observed for commercial utility that the amounts of wood extractives after fungal treatment (OF Pile Pulp or OPC Pile Pulp) did not decrease relative to the Wet Pile pulp, nor from the normal mill wood extractives of pulp data as collected from May '99-February 2000. Therefore, contrary to the laboratory results of the fungi (Example 4 showing resin component degradation by the organisms of choice), in the true setting of the utility of the fungi the resin component numbers are not decreased from the controls or normal mill running without fungal use.

&null;0136&null; BRIGHTNESS: Brightness's of the pulps from the fungal treated pulps showed that the OPC pile pulp from the TWWP had 54.0% ISO brightness, and after bleaching 78.3% ISO brightness. The OF pile, which during the growth assays showed significant growth of the albino O. floccosum, and less growth of staining species showed improved brightness with the OF pip from the TWWP having a brightness of 66.8% ISO brightness and after bleaching a 85.5% ISO brightness, a significant improvement in brightness.

7

TABLE 7

BLEACHING RESULTS:

O. floccosum

O. piceae

Mill seasoning

Caustic

66.8%

54.0%

&null;&null;59%

extracted

Bleached

85.5%

78.3%

80-82%

&null;0137&null; Finally a further experiment comprising inoculating 6000 tons of fungi with O. floccosum 13 and 40 was performed. In processing the pulp through chemical pulping after the fungal incubation, the mill again experienced an upward brightness shift of approximately 5 points. The pulp was processed through the mill without any problems.

&null;0138&null; The other unexpected benefit which became clear during the extended trial was a Kappa shift downwards of 5 points. This result may result in a huge cost saving as it means the mill can do more for less with regard to the cooking liquor charge on wood. Cooking liquor is one of the major manufacturing expenses. This benefit of the 5 point downwards shift of kappa (the kappa test is made from the reaction of pulp with acidic permanganate solution and the calculated number resulting is indicative of the amounts of non-cellulosic components (especially lignin) residing in the pulp, used in mill control work to indicate the degree of delignification occurring during cooking and the chemical requirement for bleaching (G. A. Smook, Handbook for Pulp & Paper Technologists)) to pulp mills is a significant achievement of increased chemical pulping efficiency. The increased pulping efficiency has been suggested in a research publication (M B Wall, G Stafford, Y Noel, A Fritz, S Iverson, R Farrell, Treatment with O. piliferum Improves Chemical Pulping Efficiency, presented at the 6th international Conference an Biotechnology in the Pulp and Paper Industry, Vienna, Jun. 11-15, 1995), but this publication showed in the laboratory only a downward shift of 1.25 kappa points and this work has never been demonstrated in a mill, and in a true commercial setting. The increased chemical pulping efficiency described here has practical significance to mills in several ways, including but not limited to, decreased amount of cooking liquor to be added to fungal treated wood chips to reach equivalent kappa (lignin) content at the same amount of cellulosic content, and as measured at the same viscosity, greater yield of pulp from equivalent cooking liquor as the cooking liquor works with greater efficiency to remove lignin and not cellulosic contents, improved pulp and fibre properties as the reduced cooking liquor does not act as much non-specifically on cellulosic components, improved benefit to the environment because less cooking liquor, and less concomitant effluent, to be used, and reduced cooking time as the cooking liquor is working with greater efficiency on fungal treated wood chips.

Example 5

&null;0139&null; Results

&null;0140&null; The albino O. floccosum sprayed wood chips produced a brighter pulp than had been observed in the mill, and which required less bleach chemical to reach a higher brightness than the standard mill pulps.

&null;0141&null; The extractives analysis indicated that the O. floccosum pulps, after 8 weeks incubation, had less extractives than normal seasoning without sprayed fungus requiring 12-16 weeks.

Example 6

&null;0142&null; 80,000 ton wood chip experiment Our fungal of New Zealand Ophiostoma floccosum OF40 and OF13 were researched in this 80,000 ton wood chip trial. The fungal inocula were provided as freeze dried powder in vacuum-sealed bags, packaged as approximately 1000 tonnes wood chips inocula per bag of each fungus strain.

&null;0143&null; A day of inoculation was typically considered about 000 tonnes wood chips. &null;tonne refers to 1,000 kg or 2200 lb. The wood chips used for trial purposes were all green wood with no additional water added prior to inoculation.&null;

&null;0144&null; The trial was established as follows:

&null;0145&null; Stage 1: OF 40 sprayed 10,000 tons (10 days)

&null;0146&null; Stage 2: OF 40 & OF 13 for 60 days, about 60,000 tons, and

&null;0147&null; Stage 3: OF 13 at the end sprayed onto 7000 tonnes,

&null;0148&null; Specifics of the chips and the spraying inocula were as follows:

&null;0149&null; Average chip size&null;25 mm&null;25 mm&null;2 mm

&null;0150&null; Density of wood&null;350-550 (average 400 kg/cub meter) (400 micro gram/cub.millimeter)

&null;0151&null; In order to estimate in the future for inocula-loading efficiency, one chip had a surface area of 1450 square. mm, volume of 1250 cub. mm and a mass of 5 gram.

&null;0152&null; 1 tonne of wood occupies 2.5 cub.meters, & chipped has an average surface area of 290 squ.meter. Fifty tonne of wood was sprayed with 2 cub. meters Water/Fungi

&null;0153&null; i.e. 2 cubic meters of water is sprayed over an average surface area of 14500 squ.meters.

&null;0154&null; i.e. 1 cubic meters of water is sprayed over an average surface area of 7250 squ.meters.

&null;0155&null; i.e. 1 cubic centimetre is sprayed over an average surface area of 7250 squ.milimeters.

&null;0156&null; Since there were about 10,000 (10E&null;4) to (10E&null;5) colony forming units of fungi per millilitre, each square millimetre of chip surface was sprayed with 0.725 to 7.25 fungi spores.

&null;0157&null; Results of the Trial

&null;0158&null; Mill Specifics

&null;0159&null; During the trial the mill was constantly making observations regarding performance of the fungal inoculated chips. A successful outcome of the trial was decided at the beginning to have no pitch shutdowns when fungal inoculated chips were run through the mill. This was the case. There were no shutdowns at the mill because of pitch during the three months of fungal use.

&null;0160&null; Importantly, the pulp was clean leaving the mill.

&null;0161&null; Analysis Method for Fungal Trial Extractives

&null;0162&null; The samples were freeze dried and then ground on a Wiley mill to pass a 40 mesh sieve and then extracted using the Soxtec apparatus with t-buthymethyl ether as solvent. The total extractives are expressed on the basis of oven dried starting wood.

&null;0163&null; Gas Chromatography

&null;0164&null; (According to Procedure of Orsa and Holmbom, J. Pulp Pap. Sci., 20(12) J361, 1994)

&null;0165&null; The internal standard and synthetic mixture solutions were prepared. The internal standard (50 &null;L) was added to the extract vial containing approximately 0.6 mg of material, and then treated with an excess of ethereal diazomethane. The solvent and excess diazomethane were evaporated under a stream of nitrogen dichloromethane (400 &null;L) added and the vial was sealed.

&null;0166&null; A Hewlett Packard 5890 series II GC with on-column injection and FID detector was used, with 1 &null;L injected onto the column, which was a BP1, 10 m&null;0.54 mm&null;2 mm.

&null;0167&null; GC Conditions

&null;0168&null; Injector temperature&null;80&null; C. initially, held for 2 minutes and ramped to 340&null; C. at 20&null; C./minute and then held at 340&null; C. Detector temperature 340&null; C.

&null;0169&null; Oven temperature&null;40&null; C. initially, held for 2 minutes. Then it was increased to 100&null; C. at 60&null; C./minute, held for 2 minutes, ramped to 200&null; C. at 4&null; C./minute and then ramped to 340&null; C. at 5&null; C./min and held for 10 minutes.

&null;0170&null; The helium carrier gas flow rate was constant at 12 kPa.

&null;0171&null; All retention times were determined by comparison to authentic standards.

&null;0172&null; Soxtec Method

&null;0173&null; The finely ground wood (&null;2 g) was accurately weighed in duplicate into small cellulose thimbles (26 mm&null;60 mn). At the same time sample (&null;1 g) was accurately weighed in duplicate for moisture content. The thimble was put into the Soxtec apparatus and 60 mL t-butylmethyl ether added to a pre-weighed aluminium cup containing anti bumping granules. The thimble was placed in the boiling position for 30 minutes and then raised to the rinsing position for 60 minutes. After this time, the solvent was removed by evaporation, and the last trace of solvent removed under vacuum. The dry cup was re-weighed and the % extract calculated.

&null;0174&null; GC Chromatography

&null;0175&null; (a) Extract Preparation

&null;0176&null; The extract was washed into a volumetric flask with small volumes of acetone, and made up to the mark. An aliquot was removed to yield approximately 0.6 mg of material and transferred to a vial. The acetone was removed by a gentle stream of nitrogen, and the vial flushed with nitrogen prior to sealing. The vials were stored sealed under nitrogen in the freezer before analysis.

&null;0177&null; (b) Solutions

&null;0178&null; The internal standard solution contained pentadecanoic acid (0.8472 mg/mL), betulin (0.7782 mg/mL), cholesteryl heptadecanoate (0.94 mg/mL) and dipalmitoyloleoyl glycerol (0.4154 mg/mL) in dichloromethane. The pentadecanoic acid is a check for complete derivitisation, and the dipalmitoyloleoyl glycerol is a check for triglyceride recovery. The betulin is used as internal standard for resin and fatty acids and the cholesteryl heptadecanoate is used as internal standard for triglycerides. The ratio of the 2 internal standards is used to monitor column performance.

&null;0179&null; A synthetic resin mixture, containing 3 fatty acids, 2 resin acids and a triglyceride was also prepared in dichloromethane to check response factors.

&null;0180&null; Percentage component is determined by adding together the areas of peaks of the gas chromatagram that fall within a retention time range. This range is determined by analysis of authentic standards. However, there is no guarantee that peaks in the range are the same as the standards. Some verification work has been done on GCMS (gas chromatography/mass spectroscopy) to check peak assignments, however this was not done for triglycerides, where the samples can not be put on the GCMS. The pitch and pulp samples especially may not actually contain triglycerides at all.

&null;0181&null; The Mill sent samples of chips every two weeks of the trial to The University of Waikato for analysis. These chips had done the following:

&null;0182&null; Chips were assayed for the type of fungi growing on them by the Waikato lab's standard mycological procedures and assessed for stains an indication of the albino fungal dominance over other microorganisms on the chips and biocontrol effect.

&null;0183&null; Chips were analysed for extractives.

&null;0184&null; Fungal Isolation from Chips

&null;0185&null; The fungal isolates from the chips used were isolated and purified on selected media, and identified by morphology and in some cases by DNA probe. The purpose of these isolations is first, to detect whether the inoculated fungi sprayed onto the chips were growing, and second, to see what else might be growing on the chips. The two albino O. floccosum strains, called OF 40 and OF13, had a reversion of melanin formation such that when growing on chips, these fungi produced light brown synnema hairs that stick up from the chip. The hyphae of the fungi are non-melanised i.e. non-staining. The brown synnema proved useful for the mill and lab to be able to see when the albino was well growing on the chip. The data given in Table 9 is from all the research mill trials.

8

TABLE 9

Identification of Fungi from Chips Isolates from 800 tonnes wood chips

Mill Expt. (Nov '1999) Piles: Wet Pile (water), Dry Pile (not sprayed),

O. piceae pile and O.floccosum pile

No

Site From

ID

1

Wet pile

O. querci

2

Wet pile

O. querci

3

Dry pile

Mucor

4

Wet pile

Graphium sp. C

5

Wet pile

Graphium sp. C

6

Wet pile

O. querci

7

O. piceae

O. floccosum/Graphium sp. C

pile

8-31

O. floccosum

O. floccosum albino O. floccosum/Graphium sp. C,

pile

O. querci, Pesotum sp. C and Y

&null;0186&null; Isolates from 6000 tonnes wood chips Mill Expt. (March 2000). Pile sprayed with 50:50 mixture of O. floccosum 13 and O. floccosum 40

9

No

ID

1A

Mucor

2B

O. floccosum albino

3C

Mucor

4D

O. floccosum albino

5E

O. floccosum

6F

O. floccosum

7G

O. floccosum

2a

O. querci

2c

O. querci

2d

Mucor

2e

O. querci

7f

O. floccosum Pesotum sp Y

Isolates from 80,000 tonnes wood chips Mill Expt. (August, 2000-February 2001). Pile sprayed with 50:50 mixture of O. floccosum 13 and O. floccosum 40 or each alone

&null;0187&null; F40 Treatment

&null;0188&null; 0 week: Chips were clean, no stained chips observed.

&null;0189&null; The microorganisms that grew on media 4 and 6 after 3-6 days incubation are: A,niger, Trichoderma, Penicillium, White fungi, Bacteria, Yeast. The albino O40 with brown synnema (O. floccosum) dominated on plates media 6.

&null;0190&null; 2 week: Chips were clean, no stain, Albino O40 with Brown synnema can be seen under microscope/naked eyes.

&null;0191&null; After 3-6 day incubation on media 4/6: Mucor, Bacteria, and Albino O.floccosum (Brown synnema) dominated on media 6.

&null;0192&null; 4 week and 6 week; chips were clean no stain at 4 weeks, but at 6 weeks some stained chips observed; After 3-6 day incubation of 4/6 week samples : Mucor (fungus with an obvious Black dot), Bacteria, Trichoderma, Albino O. floccosum (brown synnema) and darker O. floccosum dominated on media 6.

&null;0193&null; 8 and 10 week samples: Chips little bit darker, Albino O. floccosum brown synnema fungi growing well and black dot fungi can be seen under microscope After 3-6 day incubation on media 4/6: Mucor, Rhizopus, bacteria yeast and albino O. floccosum were dominated on media 6.

&null;0194&null; F13&null;F40 Treatment

&null;0195&null; 2 week: Chips were clean no stain. Brown synnema on chips can be seen under microscope. After 3-6 days on media 4/6, Mucor, Rhizopus, Bacteria and Albino brown synnema (O. floccosum) on media 6.

&null;0196&null; 4 and 6 Week Sample:

&null;0197&null; 4 week sample still clean, no stained chips, but on 6 week sample chips become darker. Brown synnema and black hairy fungi cm be seen under microscope.

&null;0198&null; On media 4/6: Mucor, Trichoderma bacteria yellowish hypae fungi, albino O. floccosum and darker O. floccosum.

&null;0199&null; 8 and 10 week. Some chips were darkened. Black dot fungi on chips and albino O. floccosum growing well.

&null;0200&null; After 3-6 days incubation on media 4/6. Mucor (black dot), Bacteria, Rhizophus. Albino O. floccosum dominated.

&null;0201&null; F13 Treatment

&null;0202&null; 2 week: Clean, but stain on some chips. Under microscope: black hairy fungi, Trichoderma, and albino brown synnema (O. floccosum).

&null;0203&null; Under microscope observation there were no difference between sample from surface, and bottom of pile.

&null;0204&null; After 3-6 days: Mucor, Aspergillus, brown synnema (floccosum).

&null;0205&null; 4 week: Chips look stained. Brown synnema on all chips, Black synnema, Black hairy fungi, Trichoderma, Penicillium.

&null;0206&null; After 3-6 days: Mucor, bacteria, Trichoderma. Floccosum (dominated).

&null;0207&null; 6 and 8 week sample: Chips were darkened and stained, under scope,: Brown synnema (floccosum), Trichoderma, black hairy fungi.

&null;0208&null; After 3-6 days: Mucor, bacteria, Trichoderma, Penicillium. Brown synnema/floccosum dominated.

&null;0209&null; 10 week sample: Less stain than samples 6 and 8 weeks. Under microscope observe the brown synnema on chips, after 6-8 days on media 4 and 6: Penicillium, Mucor, yeast, bacteria, Albino brown synnema (O. floccosum), black synnema (O. querci&null;)

&null;0210&null; Untreated Chips Sample

&null;0211&null; 30 week Stock pile.

&null;0212&null; Mostly clean no stain, but some chips were colonised with black hairy fungi.

&null;0213&null; 30 wk stock pile: Trichoderma, penicillium, Bacteria, Black synnema (Querci).

&null;0214&null; Chips: Black synnema (querci&null;), blach hairy fungi, Penicillium, Mucor, bacteria

&null;0215&null; Truck: Penicillium, Mucor, Yeast.

&null;0216&null; Analysis

&null;0217&null; Statistics Data from Lab and Trial Extractives Data

&null;0218&null; Tables A to L present statistical data.

&null;0219&null; Statistic extractives for Lab Trial with the parameters Strains: 1&null;F13 2&null;F40

&null;0220&null; Doses 1&null;0(control), 2&null;{fraction (1/10)}&null;, 3&null;&null;, 4&null;10&null;

&null;0221&null; Media 1&null;YM 2&null;Peptone

10

TABLE A

Analysis of Variance for % MTBE (Two-way analysis of Variance

between Strain vs doses)

Source

DF

SS

MS

F

P

Strain

1

0.3180

0.3180

4.26

0.050

Doses

3

2.1335

0.7112

9.52

&null;0.000**

Interaction

3

0.1972

0.0657

0.88

0.465

Error

24&null;

1.7922

0.0747

Total

31&null;

4.4409

**Significantly different (P<0.05) doses level significantly affected to % MTBE

&null;0222&null;

11

TABLE A

Analysis of Variance for % MTBE (Two-way analysis of Variance

between Strain Vs Media)

Source

DF

SS

MS

F

P

Strain

1

0.318

0.318

2.30

0.140

Media

1

0.250

0.250

1.81

0.189

Interaction

1

0.003

0.003

0.02

0.884

Error

28&null;

3.870

0.138

Total

31&null;

4.441

Strain, media and the interaction between them did not significantly affected to % MTBE

&null;0223&null;

12

TABLE C

Analysis of Variance for % MTBE (Two-way analysis of Variance

between Doses vs Media)

Source

DF

SS

MS

F

P

Strain

3

2.1335

0.7112

9.96

&null;0.000**

Doses

1

0.2503

0.2503

3.50

0.073

Interaction

3

0.3426

0.1142

1.60

0.216

Error

24&null;

1.7144

0.0714

Total

31&null;

4.4409

*Significantly different (P<0.05), Strain significantly affected to % MTBE

&null;0224&null;

13

TABLE D

Analysis of Variance for % Fatty Acids (Two-way analysis between

Doses and Media)

Source

DF

SS

MS

F

P

Dose

3

0.1959

0.0653

2.07

0.131

Media

1

0.0968

0.968&null;

3.06

0.093

Interaction

3

0.1080

0.0360

1.14

0.353

Error

24&null;

0.7587

0.0316

Total

31&null;

1.1594

% FA did not significantly influenced by doses, media or interaction.

&null;0225&null;

14

TABLE E

Analysis of Variance for % FA (Two-way analysis between Strain

Vs Doses)

Source

DF

SS

MS

F

P

Strain

1

0.0242

0.0242

0.70

0.412

Media

3

0.1959

0.0653

1.88

0.160

Interaction

3

0.1056

0.0352

1.01

0.404

Error

24&null;

0.8336

0.0347

Total

31&null;

1.1594

&null;0226&null;

15

TABLE F

Analysis of Variance for % Fatty acids (Two-way analysis between

Strain vs doses)

Source

DF

SS

MS

F

P

Strain

1

0.242

0.0242

0.70

0.412

Doses

3

0.1959

0.653

1.88

0.160

Interaction

3

0.1056

0.352

1.01

0.404

Error

24&null;

0.8336

0.347

Total

31&null;

1.1594

&null;0227&null;

16

TABLE G

Analysis of variance for % Resin Acids (Two-way analysis between

Strain and Doses)

Source

DF

SS

MS

F

P

Strain

1

0.0957

0.0957

3.60

0.70*

Doses

3

0.5150

0.1717

6.45

&null;0.02**

Interaction

3

0.0792

0.0264

0.99

0.413

Error

24&null;

0.6386

0.0266

Total

31&null;

1.3286

**Significantly diff (P<0.05), % RA significantly affected by doses

&null;0228&null;

17

TABLE H

Analysis of Variance for % Resin Acids (Two-way analysis between

Strain Vs Doses)

Source

DF

SS

MS

F

P

Strain

1

0.0957

0.0957

2.23

0.147

Doses

1

0.0205

0.0205

0.48

0.495

Interaction

1

0.0088

0.0088

0.20

0.655

Error

28&null;

1.2036

0.0430

Total

31&null;

1.3286

&null;0229&null;

18

TABLE I

Analysis of variance for % Resin Acids (Two-way analysis between

Doses Vs Media)

Source

DF

SS

MS

F

P

Doses

3

0.5150

0.1717

7.37

0.001&null;

Media

1

0.0205

0.0205

0.88

0.357**

Interaction

3

0.2343

0.0781

3.35

0.036*&null;

Error

24&null;

0.5587

0.0233

Total

31&null;

1.3286

**Significantly diff(P<0.5), Media affected to % RA

&null;0230&null;

19

TABLE J

Analysis of variance of % Triglycerides (Two-way

analysis between Doses Vs Media)

Source

DF

SS

MS

F

P

Doses

3

0.048838

0.016279

52.09

0.000**

Media

1

0.005512

0.005512

17.64

0.000**

Interaction

3

0.007937

0.002646

8.47

0.001**

Error

24&null;

0.007500

0.000313

Total

31&null;

0.069788

**Significantly different (P < 0.05), Doses and media affected to % TG.

&null;0231&null;

20

TABLE K

Analysis of variance of % Triglycerides (Two-way

analysis between Strain and Media)

Source

DF

SS

MS

F

P

Strain

1

0.00245

0.00245

1.11

0.300

Media

1

0.00551

0.00551

2.50

0.125

Interaction

1

0.00020

0.00020

0.09

0.765

Error

28&null;

0.06163

0.00220

Total

31&null;

0.06979

&null;0232&null;

21

TABLE L

Analysis of variance of % Triglycerides (Two-way

analysis between Strain vs Doses)

Source

DF

SS

MS

F

P

Strain

&null;1

0.002450

0.002450

4.01

0.057*

Doses

&null;3

0.048838

0.016279

26.67

0.000**

Interaction

&null;3

0.003850

0.001283

2.10

0.126

Error

24

0.014650

0.000610

Total

31

0.069788

**Significantly diff (P < 0.05), Doses and strain affected to % TG.

&null;0233&null; Definitions and Explanations

&null;0234&null; When varying the dose of the fungi, the mill trial fungal dose was called 1&null; (or one times) and in the lab this was varied to one-tenth the mill dose ({fraction (1/10)}&null;) or 10 times the mill dose (10&null;).

&null;0235&null; % MTBE means the total extractives as removed by methyl tert-butyl ether (MTBE) from the wood chips. The components of fatty acids, resin acids and triglycerides were identified on the gas chromatogram. In Table 1, for the Lab data, the terms &null;Peptone&null; and &null;YM&null; are the growth medium that the lab uses to prepare the fungi. The term &null;powder&null; means the freeze-dried powder inocula of the fungi that was provided to the mill for the 80,000 tonnes wood chip trial.

&null;0236&null; The components of radiata pine extractives that cause pitch accumulations are not known. Some suspect it might be the fatty acids and/or resin acids that cause pitch to start to accumulate. It does not appear to be the overall total extractives or the triglyceride content, but this has not been proven.

&null;0237&null; From Table 11 there are the following conclusions (following from the statistical analysis of data provided above):

&null;0238&null; Lab Results: Total Extractives (All Lab Results are 4 Week, Fungal Incubation on Chips)

&null;0239&null; 1. In the lab, the uninoculated chips of the mill (called frozen control as these chips were maintained frozen until the extractives were run on them) had sing total extractives of between 1.97% of oven dry weight of wood. Extractives of 1.97% may not seem much, but consider that of 1000 tonnes wood chips processed per day, it could represent 19.7 tonnes of extractive &null;pitch&null; (if the sulfite cook process extracted like the MTBE solvent.)

&null;0240&null; 2. In the lab, the best fungal reduction of overall extractives was to about 0.56% using OF 13 either grown freshly in peptone or as produced in the powder form for the mill trial. That represents more than two-thirds reduction of overall extractives by the fungus OF13. The best reduction for OF40 of overall extractives in the lab was reduction by little more than a half of overall extractives by the fungus.

&null;0241&null; Lab Results Fatty Acids and Resin Acids

&null;0242&null; 3. The best reduction of % fatty acids by fungi was OF40 at 10 times inocula in YM, or OF13 at 1 times inocula in peptone; 0.14% starting control fatty acids were reduced in each case to 0.02% (reduction of 86%).

&null;0243&null; 4. The best reduction of % resin acids by fungi was OF13 (with several different doses and medium) and the OF40 and OF13 powder inocula; 0.67% starting control resin acids were reduced to 0.14% (reduction of 80%).

&null;0244&null; 5. The best reduction of % triglycerides by fungi was achieved by both OF13 and OF40 and, 0.15% starting control triglycerides were reduced to 0.01% (reduction of 94%).

&null;0245&null; With both OF40 and OF13 powder, the 1&null; dosage used in the lab, same dosage as used in the mill, was better in total extractives, fatty acid, resin acid and triglycerides decrease than the 10 times, or one-tenth dosage.

&null;0246&null; Mill Results; Extractives OF 40 (Mill Results are Generally 10 Weeks Fungal Incubation on Chips)

&null;0247&null; For OF40, the 0 week chips had total extractives of 1.94% and after 2 weeks the total extractives had risen to 2.39%. Since this type of shift in total extractives makes it difficult to try to analyse what was happening, it is best to look at FIG. 2 as the overall trend in the extractives of the chips as a function of incubation time in the chip piles. In FIG. 2 it is clear that first, there was reduction of the four extractive parameters, and second the greatest reduction was after 10 weeks than at any other time point, though % MBTE Total extractives and % triglycerides have decreased to their lowest levels by 8 weeks.

&null;0248&null; For the chips inoculated with both OF40 & OF13, see FIG. 3, it appears at 6 weeks that there was a peak of &null;resin&null; extractives. Overall again the numbers decrease by the time 10 weeks incubation is achieved, but not as much reduced as with OF13 alone, though very close.

&null;0249&null; As the data of the OF13 will not be completed until end of March we have to wait until then to understand by these parameters what is the most effective fungal inoculation. Again the caution is that no one knows the extractive parameters that cause pitch shutdowns and they may not be one of the four criteria tested in this analysis.

&null;0250&null; The pulps resulting from the OF40 and OF40& OF13 combined inoculated and incubated chips were analysed also for extractives, given in Table 11 and graphed in FIG. 4 and 5.

&null;0251&null; From this data, the OF40 pulp at the Chemiwasher stage still contains a significant amount of extractives The chips of OF40 at 10 weeks had 1.16% MBTE total extractives, and after the sulfite cook, the pulp at the Chemiwasher has 0.92% MBTE&null;in other words the cook only removed 20% of the total extractives. This pulp mainly contained resin acids, (20% less resin acids than were in the chips) and the fatty acids and triglycerides in the pulps were about the same amounts as present in the chips, but one-seventh and one-twentieth the amounts respectively as the amount of resin acids present in the pulps. Resin acid is a bigger component of extractives in chemiwasher pulp than anything else we tested. Note that the components of resin acids, fatty acids and triglycerides are only about half of the total extractives&null;the other half is not identified.

&null;0252&null; The data of the pulps resulting from the OF40& OF13 combined inoculated chips follows the same trends as the OF 40 pulps, though there is 7% more total extractives in the OF40& OF13 chemiwasher pulp and 11% less resin acid in the OF40 & OF 13 chemiwasher pulp than the OF40 chemiwasher pulp. Again, it looks like resin acids are more present in the chemiwasher pulp than the other components identified, and there is little extractive in the other pulps.

&null;0253&null; The analysis of the OF13 & Of40 pitch deposit showed that 89% of the pitch deposit was MBTE solvent exactable. Of the extractables we have identified only about one-third of the components. The deposit contains three times the amount of fatty acid as resin acids, and not much triglycerides. That deposited pitch has 22% fatty acids versus 7% resin acids.

&null;0254&null; Lipase Assay on Trial Organisms

&null;0255&null; Fungi, in order to colonise wood effectively, have to be able to secrete specific enzymes to readily metabolise the essential compounds. One such extracellular enzyme, lipase is particularly important in this study to understand better the mechanism of resin degradation. Lipase catalyses the hydrolysis of triglycerides to glycerol, fatty acids and resin acids. Below is a brief outline of a lipase assay which uses p-nitrophenyl palmitate (PNP) as a substrate. An enzyme unit is that amount of enzyme that will cause release of 1 &null;mole of PNP per minute under assay conditions.

&null;0256&null; The fungi used in this assay were isolated off the 8 weeks incubated wood chips, inoculated with either OF40, OF40&13 or OF13. Overnight cultures of the fungi were standardised to 107 cells/ml and inoculated into Peptone and YM media

&null;0257&null; Assay

&null;0258&null; Substrate p-nitrophenyl palmitate; Assayed at 40&null; C. for 20 minutes.

&null;0259&null; Cooled on ice and reaction stopped with 0.1 M Na2CO3&null;10% Triton.

&null;0260&null; Absorbance read at 400 nm. Activity: &null;A400/0.011&null;10/Time&null;mU/ml

22

Lipase Activity

(mU/ml/107 cells)

Organism

Peptone Media

YM Media

8 weeks samples

467

131

(Mixture of O. floc 40 & O. floc. 13)

O. floccosum 40

475

107

O. floccosum 13

365

62&null;

&null;0261&null; It can be concluded that these strains of Ophiostoma floccosum, as isolated of the wood chips, produced a very significant amount of lipase enzyme, This explains its ability to invade sapwood so effectively and to be able to use resin as a nutrient source. The University of Waikato lab is now looking for other resin degrading enzymes to remove more of the resin components.

Example 6

&null;0262&null; Results

&null;0263&null; 1. In the lab, looking at the statistics of the extractives decrease, strain, media and the interaction between them did not significantly affected to % MTBE

&null;0264&null; 2. Use of fungi at the mill went smoothly with fungi inocula sprayed evenly to inoculate wood chips as conveyed by the system as evidenced by the good growth of the albino fungi on chips assayed from 2 weeks chip pile incubation to 10 weeks incubation.

&null;0265&null; 3. The mill had no shutdowns when using the fungal inoculated chips with less seasoning than routinely done.

&null;0266&null; References

&null;0267&null; Farrell, R. L., Blanchette, R. A., Brush, T. S., Hadar, Y., Iverson, S., Krisa K., Wedler, P. A., Zimmerman, W. (1993). Cartpip TM: A biopulping product for control of pitch and resin acid problems in pulp mills. J. Biotechnol. 30: 115-122.

&null;0268&null; Farrell R. L., Blanchette, R. A., Brush, T. S., Gysin, B., Hadar, Y., Perollaz, J. J., Wendler, P. A., and Zimmerman W. &null;Cartapip&null;&null; A Biopulping Product for Control of Pitch and Resin Acid problems in Pulp Mills in Biotechnology in the Pulp and Paper Industry, Kyoto, Japan Editor: M. Kuwahara and M. Sbimada, Uni Publishers Co., Ltd., Tokyo, Japan 1992, p. 27-32.

&null;0269&null; Hoffmann, G. C., Brush, T. S., Farrell, R. L., &null;A Biopulping Product for Control of Pitch and Resin Acid Problems in Pulp and Paper Products&null;, Naval Stores Review, 102(3) 10-12 (1992).

&null;0270&null; Kay, S. J., Farrell, R. L., Hadar, E., Hadar, Y., Blanchette, R. A., Harrington, T. C. (1997). Sapstain in New Zealand&null;the cause and a potential anti-sapstain solution. Proceedings on the 11th Biennial Conference of the Australasian Plant Pathology Society, pp.21.

&null;0271&null; Wendler, P. A., Brush, T. S., Iverson, S., Krisa, K., Zimmerman, W., and Farrell, R. L., &null;Biological Control of Pitch Problems in Paper Mills&null;, Kemia Kemi 19, 262-264 (1992).

&null;0272&null; White-McDougall, W. J., Blanchette, R. A., Farrell, R. L. (1998). Biological control of the stain fungi on Populus tremuloides using selected Ophiostoma isolates. Hozforschung, 52(3), 234-240.

&null;0273&null; Behrendt, C. J., R. A. Blanchette and R. L. Farrell 1995. An integrated approach, using biological and chemical control, to prevent blue stain in pine logs. Can J. Bot. 73:613-619.

&null;0274&null; Behrendt C J, Blanchette, R A and Farrell, R L. (1995) Biological Control of Blue Stain Fungi in Wood: Investigation of Fungal Interactions in the Laboratory and Field. Phytopathology 85, 92-97.

&null;0275&null; Zimmerman, W. C., Blanchette, R. A., Bumes, T. A., Farrell, R. L. (1993). Melanin and perithecial development in Ophiostoma piliferum. Mycologia 87. 857-863.

&null;0276&null; Wall, M. B., Stafford, G., Noel, Y., Fritz, A., Iverson, S., Farrell, R. (1995) Treatment with O. piliferum Improves Chemical Pulping Efficiency; 6th International Conference on Biotechnology in the Pulp and Paper Industry