Leaf gas exchange and chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence of <Emphasis Type="Italic">Eucalyptus urophylla</Emphasis> in response to <Emphasis Type="Italic">Puccinia psidii</Emphasis> infection

One of the most important diseases of eucalyptus plantations is caused by the rust fungus Puccinia psidii. While the genetic basis of rust resistance has been addressed recently, little is known about the physiological aspects of Eucalyptus–P. psidii interaction. In order to fill this gap, we undertook a study investigating the effects of P. psidii infection on photosynthetic processes of two E. urophylla clones with contrasting resistance to the pathogen. Our results show that gas exchange and chlorophyll a fluorescence parameters were virtually unaffected in the resistant clone. In the susceptible clone, photosynthetic rates were chiefly constrained by biochemical limitations to carbon fixation. Photosynthesis was impaired only in symptomatic tissues since the reductions in photosynthetic rates were proportional to the diseased leaf area. Rust infection provoked chronic photoinhibition to photosynthesis in the susceptible clone. Overall, differences in the ability for light capture, use and dissipation may play a significant role in explaining the clonal differences in Eucalyptus in response to P. psidii infection. To our knowledge, this is the first report of the effect of rust infection on gas exchange and chlorophyll a fluorescence parameters in Eucalyptus.     

Chitinase isozymes induced by TYMV and <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> during compatible and incompatible interaction with <Emphasis Type="Italic">Brassica napus</Emphasis>

Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Microscopy and proteomic analysis of the non-host resistance of <Emphasis Type="Italic">Oryza sativa</Emphasis> to the wheat leaf rust fungus, <Emphasis Type="Italic">Puccinia triticina</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>

Rice (Oryza sativa) cv. Nipponbare expresses non-host resistance (NHR) to the wheat leaf rust fungus, Puccinia triticina f. sp. tritici (Ptt). When the leaves of cv. Nipponbare were inoculated with Ptt, approx 93% of the urediniospores germinated on the leaf surface, but only 10% of the germinated spores formed appressoria over the stomata at one day post inoculation (1 dpi). Hydrogen peroxide (H₂O₂) accumulated in host cells around the appressoria at 3 dpi. Approx. 3% of the appressoria produced short hyphae inside the leaf, and fluorescence was observed in tissue invaded by the hyphae by 7 dpi. At 22 dpi, 0.2% of the sites with appressoria formed branching infection hypha in mesophyll cells, but no substomatal vesicles, haustorial mother cells or haustoria were observed. Proteins were extracted from leaves 3 dpi and analyzed by two-dimensional gel electrophoresis (2-DE). A total 33 spots were reproducibly up-regulated and 9 were down-regulated by infection compared to the water inoculated control. Of these, 30 were identified by MALDI-TOF Mass Spectrometry. The identified proteins participate in defense/stress responses, energy/carbohydrate metabolism, oxidation–reduction processes, protein folding/turnover/cleavage/degradation, signal transduction and cell death regulation. The results indicates that NHR of rice to Ptt is consistent with a shift in protein and energy metabolism, increased antimicrobial activities, possibly including phytoalexin accumulation and cell wall reinforcement, increased cell repair, antioxidive and detoxification reactions, and enhanced prevention of plant cell death. Nearly half of the up-regulated identified proteins were associated with chloroplast and mitochondrial physiology suggesting important roles for these organelles during NHR.     

A comparative analysis of the development and quality of nursery plants derived from somatic embryogenesis and from seedlings for large-scale propagation of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Plants of Coffea arabica L. derived via somatic embryogenesis, namely, somaclones, were evaluated with C. arabica seedlings grown in the nursery. At the time of their transfer to the nursery, somaclones of C. arabica cvs. Caturra and Costa Rica 95 (Catimor) were smaller and less vigorous than seedlings of the same cultivars. Following an initial slow growth for a period of 10 weeks, somaclones began to grow faster than seedlings until both groups of plants were equal in size at 21 weeks (entire duration of growth in the nursery). Comparisons of aerial and root systems of 30-cm long somaclones and seedlings of two cultivars revealed that plants of somaclones were more vigorous than seedlings, based on the higher number of leaves (13–16 vs. 9), larger leaf area (1060–1280 vs. 730–890 cm²), and greater dry weight of aerial organs (8.5–12 vs. 7.0–7.5 g). For cv. Caturra, the root dry weight of somaclones was significantly greater than that of seedlings (2.7 vs. 1.9 g) and was attributable to the large diameter roots (>0.5 mm). Analysis of 176,000 F₁ hybrid somaclones revealed that these exhibited more heterogeneous growth than did the seedlings derived from zygotic embryos; moreover, there was a genotype effect. Almost 9–20% of somaclones required an additional 3–4 months of growth in the nursery, and 8–12% were culled for other undesirable horticultural attributes. Only 0.10–0.23% of somaclones displayed variant phenotypes. The observed somaclone vigor in the nursery was carried over to field performance as these plants were more precocious than seedlings and yielded coffee beans 1 year earlier than seedlings.     

Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.     

New slow-rusting leaf rust and stripe rust resistance genes <Emphasis Type="Italic">Lr67</Emphasis> and <Emphasis Type="Italic">Yr46</Emphasis> in wheat are pleiotropic or closely linked

The common wheat genotype ‘RL6077’ was believed to carry the gene Lr34/Yr18 that confers slow-rusting adult plant resistance (APR) to leaf rust and stripe rust but located to a different chromosome through inter-chromosomal reciprocal translocation. However, haplotyping using the cloned Lr34/Yr18 diagnostic marker and the complete sequencing of the gene indicated Lr34/Yr18 is absent in RL6077. We crossed RL6077 with the susceptible parent ‘Avocet’ and developed F₃, F₄ and F₆ populations from photoperiod-insensitive F₃ lines that were segregating for resistance to leaf rust and stripe rust. The populations were characterized for leaf rust resistance at two Mexican sites, Cd. Obregon during the 2008–2009 and 2009–2010 crop seasons, and El Batan during 2009, and for stripe rust resistance at Toluca, a third Mexican site, during 2009. The F₃ population was also evaluated for stripe rust resistance at Cobbitty, Australia, during 2009. Most lines had correlated responses to leaf rust and stripe rust, indicating that either the same gene, or closely linked genes, confers resistance to both diseases. Molecular mapping using microsatellites led to the identification of five markers (Xgwm165, Xgwm192, Xcfd71, Xbarc98 and Xcfd23) on chromosome 4DL that are associated with this gene(s), with the closest markers being located at 0.4 cM. In a parallel study in Canada using a Thatcher × RL6077 F₃ population, the same leaf rust resistance gene was designated as Lr67 and mapped to the same chromosomal region. The pleiotropic, or closely linked, gene derived from RL6077 that conferred stripe rust resistance in this study was designated as Yr46. The slow-rusting gene(s) Lr67/Yr46 can be utilized in combination with other slow-rusting genes to develop high levels of durable APR to leaf rust and stripe rust in wheat.     

A novel Robertsonian translocation event leads to transfer of a stem rust resistance gene (<Emphasis Type="Italic">Sr52</Emphasis>) effective against race Ug99 from <Emphasis Type="Italic">Dasypyrum</Emphasis><Emphasis Type="Italic">villosum</Emphasis> into bread wheat

Stem rust (Puccinia graminis f. sp. tritici Eriks. & E. Henn.) (the causal agent of wheat stem rust) race Ug99 (also designated TTKSK) and its derivatives have defeated several important stem rust resistance genes widely used in wheat (Triticum aestivum L.) production, rendering much of the worldwide wheat acreage susceptible. In order to identify new resistance sources, a large collection of wheat relatives and genetic stocks maintained at the Wheat Genetic and Genomic Resources Center was screened. The results revealed that most accessions of the diploid relative Dasypyrum villosum (L.) Candargy were highly resistant. The screening of a set of wheat–D. villosum chromosome addition lines revealed that the wheat–D. villosum disomic addition line DA6V#3 was moderately resistant to race Ug99. The objective of the present study was to produce and characterize compensating wheat–D. villosum whole arm Robertsonian translocations (RobTs) involving chromosomes 6D of wheat and 6V#3 of D. villosum through the mechanism of centric breakage-fusion. Seven 6V#3-specific EST–STS markers were developed for screening F₂ progeny derived from plants double-monosomic for chromosomes 6D and 6V#3. Surprisingly, although 6D was the target chromosome, all recovered RobTs involved chromosome 6A implying a novel mechanism for the origin of RobTs. Homozygous translocations (T6AS·6V#3L and T6AL·6V#3S) with good plant vigor and full fertility were selected from F₃ families. A stem rust resistance gene was mapped to the long arm 6V#3L in T6AS·6V#3L and was designated as Sr52. Sr52 is temperature-sensitive and is most effective at 16°C, partially effective at 24°C, and ineffective at 28°C. The T6AS·6V#3L stock is a new source of resistance to Ug99, is cytogenetically stable, and may be useful in wheat improvement.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> with thaumatin II gene to control fungal diseases

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l⁻¹ kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l⁻¹ kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation.     

Antisense suppression of thioredoxin<Emphasis Type="Italic">h</Emphasis>mRNA in <Emphasis Type="Italic">Brassica napus cv.</Emphasis>

In Brassica, the thioredoxinhproteins, THL1 and THL2, were previously found to be potential inhibitors of the S receptor kinase (SRK) in the Brassica self-incompatibilty response. To investigate the biological roles of THL1 and THL2 in pollen–pistil interactions, the stigma-specific SLR1 promoter was used to drive antisense THL1/2 expression in Brassica napus cv. Westar. This cultivar is normally compatible, but antisense suppression of THL1/2 led to a low level constitutive rejection of all Brassica napus pollen tested. Fluorescence microscopy revealed that the pollen rejection was a typical Brassica self-incompatibility rejection response with reduced pollen adhesion, germination and pollen tube growth. In addition, Westar was found to express the SLG₁₅ and SRK₁₅ proteins which may be the target of regulation by THL1 and THL2. Thus, these results indicate that the THL1 and THL2 are required for full pollen acceptance in B. napus cv. Westar.     

Carbon nutrition of mature green orchid <Emphasis Type="Italic">Serapias strictiflora</Emphasis> and its mycorrhizal fungus <Emphasis Type="Italic">Epulorhiza</Emphasis> sp.

We studied the nutritional modes of the orchid Serapias strictiflora and its mycorrhizal fungus Epulorhiza sp. using the differences in carbon isotopic composition (δ¹³C) of C₃ orchid and C₄ maize tissues. We found that if cultivated in substrate lacking any organic compounds, the mycorrhizal extraradical mycelia (δ¹³C = −26.3 ± 0.2 ‰) developed well, despite being fully dependent on nutrition from orchid roots (δ¹³C = −28.6 ± 0.1 ‰). If the mycorrhizal fungus had additional access to and colonized decaying maize roots (δ¹³C = −14.6 ± 0.1 ‰), its isotopic composition (δ¹³C = −21.6 ± 0.4 ‰) reflected a mixture of biotrophy and saprotrophy. No statistically significant differences in δ¹³C of new storage tubers were found between Epulorhiza-associated orchids with (δ¹³C = -28.2 ± 0.1 ‰) and without access to maize roots (δ¹³C = −28.6 ± 0.2 ‰). We conclude that autotrophy is the predominant nutritional mode of mature S. strictiflora plants and that they supply their mycorrhizal fungus with substantial amount of carbon (69 ± 3 % of the fungus demand), even if the fungus feeds saprotrophically.     

Characterisation of a new stripe rust resistance gene <Emphasis Type="Italic">Yr47</Emphasis> and its genetic association with the leaf rust resistance gene <Emphasis Type="Italic">Lr52</Emphasis>

Two Iranian common wheat landraces AUS28183 and AUS28187 from the Watkins collection showed high levels of seedling resistance against Australian pathotypes of leaf rust and stripe rust pathogens. Chi-squared analyses of rust response segregation among F₃ populations derived from crosses of AUS28183 and AUS28187 with a susceptible genotype AUS27229 revealed monogenic inheritance of leaf rust and stripe rust resistance. As both genotypes produced similar leaf rust and stripe rust infection types, they were assumed to carry the same genes. The genes were temporarily named as LrW1 and YrW1. Molecular mapping placed LrW1 and YrW1 in the short arm of chromosome 5B, about 10 and 15 cM proximal to the SSR marker gwm234, respectively, and the marker cfb309 mapped 8–12 cM proximal to YrW1. LrW1 mapped 3–6 cM distal to YrW1 in two F₃ populations. AUS28183 corresponded to the accession V336 of the Watkins collection which was the original source of Lr52. Based on the genomic location and accession records, LrW1 was concluded to be Lr52. Because no other seedling stripe rust resistance gene has previously been mapped in chromosome 5BS, YrW1 was permanently named as Yr47. A combination of flanking markers gwm234 and cfb309 with phenotypic assays could be used to ascertain the presence of Lr52 and Yr47 in segregating populations. This investigation characterised a valuable source of dual leaf rust and stripe rust resistance for deployment in new wheat cultivars. Transfer of Lr52 and Yr47 into current Australian wheat backgrounds is in progress.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Drought-induced oxidative damage and antioxidant responses in peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) seedlings

Two cultivars of peanut (Arachis hypogaea L.) which were designated as resistant (Florispan) and sensitive (Gazipasa) according to their growth retardation under drought stress conditions were compared for their oxidative damage and antioxidant responses. Sixteen days-old peanut seedlings were subjected to PEG-6000 solutions of two different osmotic potentials; −0.4 and −0.8 MPa, and various growth parameters, photosystem II activity, changes in malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline levels, activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX) and gluthatione reductase (GR) enzymes were determined. Both cultivars exhibited water deficit at −0.8 MPa osmotic potential of PEG-6000 and H₂O₂ levels significantly increased during exposure to −0.4 MPa osmotic potential. However, H₂O₂ levels were under control in both cultivars at exposure to −0.8 MPa osmotic potential. Significant proline accumulation was observed in the tissues of cv. Florispan at −0.8 MPa osmotic potential, whereas proline accumulation did not appear to be an essential part of the protection mechanism against drought in cv. Gazipasa. No significant variation in chlorophyll fluorescence values were detected in neither of the cultivars. Enzyme activity measurements revealed that Gazipasa copes well with lesser magnitudes of drought stress by increasing the activity of mainly APX, and during harsh stress conditions, only APX maintains its activity in the tissues. In cultivar Florispan, GR activity appears to take role in lesser magnitudes of drought stress, whereas CAT and APX activities appear to be very crucial antioxidative defenses during intense stress conditions. The results indicate that, the level of proline and activities of the enzymes CAT and APX are important mechanisms for the maintenance of drought tolerance in peanut plants.     

Molecular mapping of leaf rust resistance gene <Emphasis Type="Italic">Rph14</Emphasis> in <Emphasis Type="Italic">Hordeum vulgare</Emphasis>

An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F₃ lines derived from the crosses ‘Baudin’/‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the ‘Baudin’/‘PI 584760’and ‘Ricardo’/‘PI 584760’ populations, respectively.     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai