Search

Context Insects are a major threat to crop production. They can infect, damage, and reduce agricultural yields. Accurate and fast detection of insects will help insect control. From a computer algorithm point of view, insect detection from imagery is a tiny object detection problem. Handling detection of tiny objects in large datasets is challenging due to small resolution of the insects in an image, and other nuisances such as occlusion, noise, and lack of features. Aims Our aim was to achieve a high-performance agricultural insect detector using an enhanced artificial intelligence machine learning technique. Methods We used a YOLOv3 network-based framework, which is a high performing and computationally fast object detector. We further improved the original feature pyramidal network of YOLOv3 by integrating an adaptive feature fusion module. For training the network, we first applied data augmentation techniques to regularise the dataset. Then, we trained the network using the adaptive features and optimised the hyper-parameters. Finally, we tested the proposed network on a subset dataset of the multi-class insect pest dataset Pest24, which contains 25 878 images. Key results We achieved an accuracy of 72.10%, which is superior to existing techniques, while achieving a fast detection rate of 63.8 images per second. Conclusions We compared the results with several object detection models regarding detection accuracy and processing speed. The proposed method achieved superior performance both in terms of accuracy and computational speed. Implications The proposed method demonstrates that machine learning networks can provide a foundation for developing real-time systems that can help better pest control to reduce crop damage.

Yellow tailflower mild mottle virus (YTMMV, genus Tobamovirus) was identified from wild plants of solanaceous species in Australia. Nicotiana benthamiana is a species indigenous to the arid north of Australia. N. benthamiana accession RA-4 (the lab type), which has a mutant, functionally defective, RNA-dependent RNA polymerase 1 (Rdr1) gene (Nb-Rdr1m), has played a significant role in plant virology, but little study has been done regarding responses to virus infection by other accessions of N. benthamiana. All wild-collected N. benthamiana accessions used in this study harbored wild-type Rdr1 genes (Nb-Rdr1). We compared symptoms of YTMMV infection and viral RNA load on RA-4 and nine wild-collected accessions of N. benthamiana from mainland Western Australia, an island, and the Northern Territory. After inoculation with YTMMV, RA-4 plants responded with systemic hypersensitivity and all individuals were dead 35 days postinoculation (dpi). Plants of wild-collected accessions exhibited a range of symptoms, from mild to severe, and some, but not all, died in the same period. Quantitative reverse transcription PCR revealed that the Rdr1 mutation was not a predictor of viral RNA load or symptom severity. For example, wild-collected A019412 plants carried more than twice the viral RNA load of RA-4 plants, but symptom expression was moderate. For plants of most accessions, viral RNA load did not increase after 10 dpi. The exception was plants of accession Barrow-1, in which viral RNA load was low until 15 dpi, after which it increased more than 29-fold. This study revealed differential responses by N. benthamiana accessions to infection by an isolate of YTMMV. The Rdr1 gene, whether mutant or wild-type, did not appear to influence viral RNA load or disease expression. Genetic diversity of the 10 N. benthamiana accessions in some cases reflected geographical location, but in other accessions this was not so.

We report comparisons between the complete genomic sequences of five historical Western Australian isolates of subterranean clover mottle virus (SCMoV) from 1989–2000, and an infectious clone of its 1989 isolate. Sanger Sequencing (SS) and High Throughput Sequencing (HTS), or both, were used to obtain these genomes. Four of the SCMoV isolates were sequenced by SS in 1999–2002, but re-sequenced again by HTS in 2020. The pairs of sequences obtained from these four isolates differed by only 18–59 nucleotides. This small difference resulted from the different sequencing methods, the

Genome- or gene-editing (abbreviated here as ‘GEd’) presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world’s growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Storing potato tubers at cold temperatures, either for transport or continuity of supply, is associated with the conversion of sucrose to reducing sugars. When cold-stored cut tubers are processed at high temperatures, with endogenous asparagine, acrylamide is formed. Acrylamide is classified as a carcinogen. Potato processors prefer cultivars which accumulate fewer reducing sugars and thus less acrylamide on processing, and suitable processing cultivars may not be available. We used CRISPR-Cas9 to disrupt the genes encoding vacuolar invertase (VInv) and asparagine synthetase 1 (AS1) of cultivars Atlantic and Desiree to reduce the accumulation of reducing sugars and the production of asparagine after cold storage. Three of the four guide RNAs employed induced mutation frequencies of 17–98%, which resulted in deletions, insertions and substitutions at the targeted gene sites. Eight of ten edited events had mutations in at least one allele of both genes; for two, only the VInv was edited. No wild-type allele was detected in both genes of events DSpco7, DSpFN4 and DSpco12, suggesting full allelic mutations. Tubers of two Atlantic and two Desiree events had reduced fructose and glucose concentrations after cold storage. Crisps from these and four other Desiree events were lighter in colour and included those with 85% less acrylamide. These results demonstrate that multiplex CRISPR-Cas9 technology can generate improved potato cultivars for healthier processed potato products.

Monilinia fructicola and M. laxa, causing brown rot in stone fruit (Prunus species), were first described from Western Australia in 1997. Our previous work indicated that original invasive isolates of both species were each of a single genotype. This research aims to compare isolates from the region in the present day in terms of genetic diversity, colony morphology, and virulence on fruit. Genetic diversity of 68 M. laxa and 66 M. fructicola isolates collected in 2016-2017 from five populations was measured using ISSR markers. The genetic diversity within M. fructicola was greater than that of M. laxa, having Shannon’s diversity indices of 0.50 and 0.41, respectively. Most genetic variation was present within populations for both M. fructicola (77%) and M. laxa (76%), with the rest of the variation between geographically separated populations. Genotype did not correlate closely with virulence of isolates as measured by symptom severity index (SSI) on inoculated plum fruit and colony morphology on potato dextrose agar (PDA) medium. M. fructicola isolates with wide genetic variation exhibited closely similar SSIs and one morphotype on PDA, whereas M. laxa isolates exhibited a broad range of SSIs and four different morphotypes.

Most weed species can adversely impact agricultural productivity by competing for nutrients required by high-value crops. Manual weeding is not practical for large cropping areas. Many studies have been undertaken to develop automatic weed management systems for agricultural crops. In this process, one of the major tasks is to recognise the weeds from images. However, weed recognition is a challenging task. It is because weed and crop plants can be similar in colour, texture and shape which can be exacerbated further by the imaging conditions, geographic or weather conditions when the images are recorded. Advanced machine learning techniques can be used to recognise weeds from imagery. In this paper, we have investigated five state-of-the-art deep neural networks, namely VGG16, ResNet-50, Inception-V3, Inception-ResNet-v2 and MobileNetV2, and evaluated their performance for weed recognition. We have used several experimental settings and multiple dataset combinations. In particular, we constructed a large weed-crop dataset by combining several smaller datasets, mitigating class imbalance by data augmentation, and using this dataset in benchmarking the deep neural networks. We investigated the use of transfer learning techniques by preserving the pre-trained weights for extracting the features and fine-tuning them using the images of crop and weed datasets. We found that VGG16 performed better than others on small-scale datasets, while ResNet-50 performed better than other deep networks on the large combined dataset., The paper is accepted by Crop and Pasture Science journal (https://www.publish.csiro.au/CP/justaccepted/CP21626)

Summary Conditions required for the combined culture of either Glomus caledonium (Nicol. &Gerd.) Trappe &Gerdemann or Glomus mosseae (Nicol. &Gerd.) Gerdemann &Trappe with suspension-cultured plant cells have been investigated. Sucrose levels (0.05 to 0.5%, w/v) lower than those used for growth of plant cells were optimal for hyphal growth of both G. caledonium and G. mosseae. In vitro hyphal growth from chlamydospores of G. caledonium was stimulated by addition of cells of wheat (Triticum aestivum L. cv. Maris Butler), lucerne (Medicago sativa L. cv. Europ) and potato (Solanum tuberosum L. cv. Maris Piper). The presence of wheat cells similarly stimulated hyphal growth from chlamydospores of G. mosseae. Further tests on the effect of lucerne cells on G. caledonium indicated that a volatile substance was involved in the improvement of hyphal growth.

In 1997, Monilinia fructicola and Monilinia laxa, fungi causing brown rot disease in stone fruit (Prunus species), were identified from Western Australia for the first time. Up until then, Monilinia were quarantine species, and importation of stone fruits to W.A. was prohibited. After Monilinia was identified in W.A., importation of stone fruit from sources outside W.A. was progressively permitted. Today, Monilinia is present in all stone fruit production regions in W.A. The aim of this study was to determine if the genotypes responsible for the first incursion subsequently spread, or if new genotypes have since become established. ISSR markers were used to identify the genotype of isolates collected during the initial incursion event in 1997, and compare them with isolates collected subsequently. Eight M. fructicola genotypes were identified, including a monotypic one on a fresh peach imported from the USA. M. fructicola isolates collected during the initial incursion in 1997 and an isolate from cherry collected in South Australia in the same year were all of the same genotype, suggesting fruit or germplasm from S.A. as the source of the W.A. incursion. However, this incursion genotype appears not have persisted, with different genotypes subsequently becoming widely or locally established. Four genotypes of M. laxa were identified. In contrast to M. fructicola, the 1997 incursion genotype of M. laxa has become widely established in W.A., infecting both stone fruits and pome fruits.

Control of plant-parasitic nematodes (PPNs) via host-induced gene silencing (HIGS) involves rational selection of genes and detailed assessment of effects of a possible knockdown on the nematode. Some genes by nature may be very important for the survival of the nematode that knockdown may be resisted. Possible silencing and effects of 20 such genes involved in the RNA interference (RNAi) pathways of Meloidogyne incognita were investigated in this study using long double-stranded RNAs (dsRNAs) as triggers. Two of the genes, ego-1 and mes-2, could not be knocked down. Expression of six genes (xpo-1, pash-1, xpo-2, rha-1, ekl-4, and csr-1) were significantly upregulated after RNAi treatment whereas for 12 of the genes, significant knockdown was achieved and with the exception of mes-2 and mes-6, RNAi was accompanied by defective phenotypes in treated nematodes including various degrees of paralysis and abnormal behaviors and movement such as curling, extreme wavy movements, and twitching. These abnormalities resulted in up to 75% reduction in infectivity of a tomato host, the most affected being the J2s previously treated with dsRNA of the gfl-1 gene. For 10 of the genes, effects of silencing in the J2s persisted as the adult females isolated from galls were under-developed, elongated, and transparent compared to the normal saccate, white adult females. Following RNAi of ego-1, smg-2, smg-6, and eri-1, reduced expression and/or the immediate visible effects on the J2s were not permanent as the nematodes infected and developed normally in tomato hosts. Equally intriguing was the results of RNAi of the mes-2 gene where the insignificant change in gene expression and behavior of treated J2s did not mean the nematodes were not affected as they were less effective in infecting host plants. Attempt to silence drsh-1, mut-7, drh-3, rha-1, pash-1, and vig-1 through HIGS led to reduction in nematode infestation by up to 89%. Our results show that genes may respond to RNAi knockdown differently so an exhaustive assessment of target genes as targets for nematode control via RNAi is imperative.

Endophytic fungal colonization may influence how plants respond to environmental stress. Two promising fungal isolates, one resembling Cladosporium cladosporioides and another unidentified ascomycetous fungus, isolated from wild N. benthamiana plants in northern Australia were inoculated to plants of the research accession of N. benthamiana (RA-4). Inoculated seedlings were grown under adequate or water deficit conditions. We examined leaf metabolites using gas chromatography-mass spectrometry (GC–MS) to compare levels of sugars, sugar alcohols, amino acids and other metabolites at various stages of plant growth and stress application. Ninety-three metabolites were detected in leaves, including 20 sugars, 13 sugar alcohols, 21 amino acids, 29 organic and fatty acids and ten other compounds. Endophyte colonization caused significantly differential accumulation of 17–21 metabolites when the plants were grown under well-watered condition. The presence of endophytes under water stress conditions caused differential accumulation of cytosine, diethylene glycol, galactinol, glycerol, heptadecanoate, mannose, oleic acid, proline, rhamnose, succinate, and urea. Accumulation of these metabolites suggests that fungal endophytes influence plants to accumulate certain metabolites under water-stress. Further, plants colonised by the two different endophytes tested, showed some differences in the metabolites they accumulated. Colonization with endophytic fungi significantly increased root dry mass and relative water content in plants under severe water stress condition, suggestive of a symbiotic relationship between these fungi and N. benthamiana plants, a species adapted to the hot and unpredictable soil moisture conditions of northern Australia. We reveal that endophyte colonization triggers reprogramming of host metabolism and indices changes in host development. This study sheds lights on the mechanisms underlying increased tolerance to water stress in plants conferred by fungal endophytes. Fungal endophytes have the potentials for application to increase the inherent water stress tolerance of crops.

Small grain cereals have served as the basis for staple foods, beverages, and animal feed for thousands of years. Wheat, barley, oats, rye, triticale, rice, and others are rich in calories, proteins, carbohydrates, vitamins, and minerals. These cereals supply 20% of the calories consumed by people worldwide and are therefore a primary source of energy for humans and play a vital role in global food and nutrition security. Global production of small grains increased linearly from 1960 to 2005, and then began to decline. Further decline in production is projected to continue through 2050 while global demand for these grains is projected to increase by 1% per annum. Currently, wheat, barley, and oat production exceeds consumption in developed countries, while in developing countries the consumption rate is higher than production. An increasing demand for meat and livestock products is likely to compound the demand for cereals in developing countries. Current production levels and trends will not be sufficient to fulfill the projected global demand generated by increased populations. For wheat, global production will need to be increased by 60% to fulfill the estimated demand in 2050. Until recently, global wheat production increased mostly in response to development of improved cultivars and farming practices and technologies. Production is now limited by biotic and abiotic constraints, including diseases, nematodes, insect pests, weeds, and climate. Among these constraints, plant-parasitic nematodes alone are estimated to reduce production of all world crops by 10%. Cereal cyst nematodes (CCNs) are among the most important nematode pests that limit production of small grain cereals. Heavily invaded young plants are stunted and their lower leaves are often chlorotic, forming pale green patches in the field. Mature plants are also stunted, have a reduced number of tillers, and the roots are shallow and have a “bushy-knotted” appearance. CCNs comprise a number of closely-related species and are found in most regions where cereals are produced.

In 2016 and 2017, 90 fungal isolates were collected from Prunus species exhibiting symptoms of brown rot disease at 12 sites in stone fruit production areas in Western Australia. ITS region analysis showed that 49 isolates belonged to Monilinia laxa and 34 to M. fructicola, species that cause brown rot in stone fruit. The two species were spatially separated to the south of the Perth Hills region, where only M. laxa was found, and to the north of Perth Hills where only M. fructicola was found. The two species co-existed only in the Perth Hills. The implications for control and trade are discussed, as is the need to implement biosecurity guidelines to prevent mixing of the two species where currently only one exists.

Control of plant-parasitic nematodes (PPNs) via host-induced gene silencing (HIGS) involves rational selection of genes and detailed assessment of effects of a possible knockdown on the nematode. Some genes by nature may be very important for the survival of the nematode that knockdown may be resisted. Possible silencing and effects of 20 such genes involved in the RNA interference (RNAi) pathways of Meloidogyne incognita were investigated in this study using long double-stranded RNAs (dsRNAs) as triggers. Two of the genes, ego-1 and mes-2, could not be knocked down. Expression of six genes (xpo-1, pash-1, xpo-2, rha-1, ekl-4, and csr-1) were significantly upregulated after RNAi treatment whereas for 12 of the genes, significant knockdown was achieved and with the exception of mes-2 and mes-6, RNAi was accompanied by defective phenotypes in treated nematodes including various degrees of paralysis and abnormal behaviors and movement such as curling, extreme wavy movements, and twitching. These abnormalities resulted in up to 75% reduction in infectivity of a tomato host, the most affected being the J2s previously treated with dsRNA of the gfl-1 gene. For 10 of the genes, effects of silencing in the J2s persisted as the adult females isolated from galls were under-developed, elongated, and transparent compared to the normal saccate, white adult females. Following RNAi of ego-1, smg-2, smg-6, and eri-1, reduced expression and/or the immediate visible effects on the J2s were not permanent as the nematodes infected and developed normally in tomato hosts. Equally intriguing was the results of RNAi of the mes-2 gene where the insignificant change in gene expression and behavior of treated J2s did not mean the nematodes were not affected as they were less effective in infecting host plants. Attempt to silence drsh-1, mut-7, drh-3, rha-1, pash-1, and vig-1 through HIGS led to reduction in nematode infestation by up to 89%. Our results show that genes may respond to RNAi knockdown differently so an exhaustive assessment of target genes as targets for nematode control via RNAi is imperative.

Genetic diversity of Bean yellow mosaic virus (BYMV) was studied by comparing sequences from the coat protein (CP) and genome-linked viral protein (VPg) genes of isolates from four continents. CP sequences compared were those of 17 new isolates and 47 others already on the database, while the VPg sequences used were from four new isolates and 10 from the database. Phylogenetic analysis of the CP sequences revealed seven distinct groups, six polytypic and one monotypic. The largest and most genetically diverse polytypic group, which had intragroup diversity of 0.061 nucleotide substitutions per site, contained isolates from natural infections in eight host species. These original isolation hosts included both wild (four) and domesticated (four) species and were from monocotyledonous and dicotyledonous plant families, indicating a generalized natural host range strategy. Only one of the other five polytypic groups spanned both monocotyledons and dicotyledons, and all contained isolates from fewer species (one to four), all of which were domesticated and had lower intragroup diversity (0.019 to 0.045 nucleotide substitutions per site), indicating host specialization. Phylogenetic analysis of the fewer VPg sequences revealed three polytypic and two monotypic groupings. These groups also correlated with original natural isolation hosts, but the branch topologies were sometimes incongruous with those formed by CPs. Also, intragroup diversity was generally higher for VPgs than for CPs. A plausible explanation for the groups found when the 64 different CP sequences were compared is that the generalized group represents the original ancestral type from which the specialist host groups evolved in response to domestication of plants after the advent of agriculture. Data on the geographical origins of the isolates within each group did not reveal whether the specialized groups might have coevolved with their principal natural hosts where these were first domesticated, but this seems plausible.

Transgenic yellow lupin (Lupinus luteus L.) plants have been generated by meristem co-cultivation with Agrobacterium tumefaciens. The binary plasmid pPZBNIa contains the bar gene under the control of a CaMV 35 S promoter. The transformation method involves inoculation of embryonic axis explants with A. tumefaciens, flooding the meristem with glufosinate, and initial culture on non-selective medium. Shoots were transferred to culture medium containing 20 mg/l glufosinate. Following subculture, shoots were grafted onto non-transgenic narrow-leafed lupin (L. angustifolius L.) seedling rootstocks, or rooted in vitro. The overall transformation efficiency, as determined at the T1 generation, was 0.05%–0.75%. The transgenic nature of plants grown to the T6 generation was confirmed by phosphinothricin acetyl transferase, PCR and Southern analyses.

Pelargonium capitatum (rose pelargonium) is a plant indigenous to southern Africa, originally brought to Western Australia for its ornamental qualities. It has since become naturalized in the Southwest Australian Floristic Region, recognized for its high level of species endemism, where it is a serious invasive weed in bushlands and coastal dunes. Since P. capitatum outcompetes native species it is listed among the top 10 most important coastal weeds of the region (3). In 2008, large patches of stunted, dying, and dead P. capitatum plants were observed within a population covering coastal dunes at Woodman Point, Western Australia (GPS coordinates 32°07′40.51″S, 115°45′28.39″E). Diseased plants had small misshapen leaves in clumps that were often chlorotic or pink, shortened internodes, and exhibited phylloidy typical of infection by a phytoplasma. From August 2009 to January 2010, samples from symptomatic and asymptomatic plants were collected from the site and from plants of an asymptomatic population at another site located on the Murdoch University campus nearby. DNA was extracted from 15 samples collected from symptomatic and asymptomatic plants at the dune site and from five at the campus site. Briefly, 2 to 5 g of leaf and stem tissue was cut into 5-mm pieces and shaken overnight in 30 ml of phosphate-buffered saline buffer. Supernatant was filtered and a pellet was collected by centrifugation. After resuspension in 500 μl of extraction buffer (200 mM Tris-HCl [pH 7.5] 250mM NaCl, 25mM ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate, and 2% polyvinylpyrrolidone), DNA was precipitated in 500 μl of cold isopropanol. Samples were tested for the presence of phytoplasma ribosomal 16S DNA by nested PCR using phytoplasma universal primers P1/P7 followed by amplification with primers Tint, R16mF2, and R16mR1 (1,2,4). Phytoplasma-specific DNA sequences were synthesized directly from amplicons using the above primers. Phytoplasma was detected from both symptomatic and asymptomatic plant samples collected from the dune site but not from the campus site. Analysis of the nine sequences obtained (GenBank Accession Nos. HM583339, HM583340, HM583341, HM583342, HM583343, HM583344, HM583345, HM583346, and HM583347) revealed high sequence identity between isolates (~99%) and with the ‘Candidatus Phytoplasma aurantifolia’ (16SrII) group of phytoplasmas (1,4). Presence of phytoplasma in symptomatic plants was confirmed by histological examination of stem sections stained with Dienes' stain. This finding is significant because there is potential for utilizing this phytoplasma to control P. capitatum where it has invaded ecologically significant sites, although its effect on indigenous plants must be determined first. Although phytoplasmas within the 16SrII group have been identified in Australia previously (1,4), to our knowledge, this is the first report of it infecting P. capitatum. References: (1) K. S. Gibb et al. Phytopathology 85:169, 1995. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) B. M. J. Hussey et al. Western Weeds. A Guide to the Weeds of Western Australia. 2nd ed. Plant Protection Society of Western Australia, Victoria Park, 2007. (4) M. Saqib et al. J. R. Soc. West. Aust. 90:175, 2007.

The root lesion nematode Pratylenchus zeae, a migratory endoparasite, is an economically important pest of major crop plants (e.g. cereals, sugarcane). It enters host roots, migrates through root tissues and feeds from cortical cells, and defends itself against biotic and abiotic stresses in the soil and in host tissues. We report de novo sequencing of the P. zeae transcriptome using 454 FLX, and the identification of putative transcripts encoding proteins required for movement, response to stimuli, feeding and parasitism. Sequencing generated 347,443 good quality reads which were assembled into 10,163 contigs and 139,104 singletons: 65% of contigs and 28% of singletons matched sequences of free-living and parasitic nematodes. Three-quarters of the annotated transcripts were common to reference nematodes, mainly representing genes encoding proteins for structural integrity and fundamental biochemical processes. Over 15,000 transcripts were similar to Caenorhabditis elegans genes encoding proteins with roles in mechanical and neural control of movement, responses to chemicals, mechanical and thermal stresses. Notably, 766 transcripts matched parasitism genes employed by both migratory and sedentary endoparasites in host interactions, three of which hybridized to the gland cell region, suggesting that they might be secreted. Conversely, transcripts for effectors reported to be involved in feeding site formation by sedentary endoparasites were conspicuously absent. Transcripts similar to those encoding some secretory-excretory products at the host interface of Brugia malayi, the secretome of Meloidogyne incognita and products of gland cells of Heterodera glycines were also identified. This P. zeae transcriptome provides new information for genome annotation and functional analysis of possible targets for control of pratylenchid nematodes.

Isolates of an Australian indigenous virus, Yellow tailflower mild mottle virus (YTMMV-Kalbarri), and an exotic virus,Pelargonium zonate spot virus (PZSV-SW13), are described from Anthocercis ilicifolia subsp. ilicifolia (red-striped tail-flower, family Solanaceae), a species endemic to Western Australia. This is the first report of either virus fromthis plant species. The complete genome sequences of YTMMV-Kalbarri and of PZSV-SW13 were obtained. YTMMV-Kalbarri shared 97% nucleotide pairwise identity with the sequence of the type isolate YTMMV-Cervantes. Thesequence PZSV-SW13 shared greatest sequence identity with the partial sequence of an Australian isolate of PZSV, alsofrom a wild plant, and with a sunflower-derived isolate of PZSV from Argentina. An experimental host range studywas done of YTMMV-Kalbarri using cultivated and wild solanaceous and non-solanaceous plants. Most solanaceousplants became systemically infected, with symptoms of systemic infection ranging from symptomless to whole plantnecrosis. Based on these studies, it is suggested that YTMMV has the potential to become a pathogen of commercialspecies of Solanaceae. This study provides further evidence that PZSV is present in wild plants in Australia, in this casean indigenous host species, and possible routes by which it invaded Australia are discussed.Keywords: anulavirus, indigenous plant virus, plant virus ecology, tobamovirus, virus invasion, virus emergenceAnthocercis (family Solanaceae, subfamily Nicotianoi-deae) is a genus of 15 plant species endemic to southernAustralia (Haegi, 1986). Previously, a tobamovirus(genus Tobamovirus, family Virgaviridae) was isolatedfrom A. littoria (yellow tailflower), a spindly shrub, 3 min height, that grows along the coastline in calcareoussand, limestone ridges and sand dunes of southwesternAustralia, but the virus was not detected from A. viscosa(sticky tailflower) plants growing in Albany, 400 km tothe south. The new tobamovirus was named Yellow tail-flower mild mottle virus (YTMMV; Wylie et al., 2014).Pelargonium zonate spot virus (PZSV; genus Anulavi-rus, family Bromoviridae) was first isolated from Pelar-gonium zonale (Geraniaceae) in Italy (Quacquarelli G Luis-Arteaga & Cambra,2000; Gebre-Selassie et al., 2002; Finetti-Sialer & Galli-telli, 2003; Liu & Sears, 2007; Escriu et al., 2009; Gulat-i-Sakhuja et al., 2009; Lapidot et al., 2010; Blouin et al.,2013; Giolitti et al., 2014). Its geographical rangeincludes much of Europe and the Americas. In Australia,PZSV was recently described for the first time in Cakilemaritima (Brassicaceae), a self-introduced exotic weedspecies (Luo et al., 2010).This study describes the complete genome sequences ofnew isolates of YTMMV and PZSV that co-infected a wildplant of Anthocercis ilicifolia subsp. ilicifolia (red-stripedtailflower), a new host species for both viruses. Althoughdetails of the host range of PZSV have been published, thepotential host range of YTMMV is not known. The natu-ral hosts of YTMMV live at the interface between wildand managed systems, and consequently the opportunityexists for the virus to expand its host range into cultivatedplant species. Thus, an experimental host range study wasundertaken of the new YTMMV isolate and the type YT-MMV isolate, and the potential of YTMMV to emerge asa pathogen of commercial importance is discussed. Fur-ther, it is speculated as to how PZSV may have enteredAustralia, given that it has not yet been detected there incommercial plantings.

Sequences representing the genomes of two distinct virus isolates infecting wild plants of two members of the genus Drakaea (hammer orchids) in Western Australia are described. The virus isolated from Drakaea livida has a bipartite genome of 4490 nt (RNA1) and 2905 nt (RNA2) that shares closest sequence and structural similarity to members of the genus Pecluvirus, family Virgaviridae, described from legumes in the Indian subcontinent and West Africa. However, it differs from pecluviruses by lacking a P39 protein on RNA2 and having a cysteine-rich protein gene located 3 0 of the triple gene block protein genes. It is the first peclu-like virus to be described from Australia. The name Drakaea virus A is proposed (DVA; proposed member of the family Virgaviridae, genus unassigned). The second virus isolate was identified from Drakaea elastica, a species classed as endangered under conservation legislation. The genome sequence of this virus shares closest identity with isolates of Donkey orchid symptomless virus (DOSV; proposed member of the order Tymovirales, family and genus unassigned), a species described previously from wild Caladenia and Diuris orchids in the same region. These viruses are the first to be isolated from wild Drakaea populations and are proposed to have an ancient association with their orchid hosts.

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive-sense ssRNA molecule of 8506 nucleotides. It is predicted to encode a single polyprotein of 314·5 kDa that is subsequently processed into three coat protein components of 13·7, 17·9 and 13·5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease and RNA-dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19–47% identity). Evidence of replication in plants was detected by RT-PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. The name Tomato matilda virus (TMaV) is proposed, and furthermore, that the genus Tomavirus (Tomato matilda virus) be created within the family Iflaviridae. This is the first report of a plant-infecting virus resembling members of the Iflaviridae.

Basic vegetative period (BVP) is an important trait for determining flowering time and adaptation to variable environments. A short BVP barley mutant is about 30 d shorter than its wild type. Genetic analysis using 557 F2 individuals revealed that the short BVP is governed by a single recessive gene (BVP-1) and was further validated in 2 090 F3 individuals. The BVP-1 gene was first mapped to barley chromosome 1H using SSR markers. Comparative genomic analysis demonstrated that the chromosome region of BVP-1 is syntenic to rice chromosome 5 and Brachypodium chromosome 2. Barley ESTs/genes were identified after comparison with candidate genes in rice and Brachypodium; seven new gene-specific markers were developed and mapped in the mapping populations. The BVP-1 gene co-segregated with the Mot1 and Ftsh4 genes and was flanked by the gene-specific markers AK252360 (0.2 cM) and CA608558 (0.5 cM). Further analysis demonstrated that barley and wheat share the same short BVP gene controlling early flowering.

The aim of this review was to undertake a survey of researchers working with plant-parasitic nematodes in order to determine a ‘top 10’ list of these pathogens based on scientific and economic importance. Any such list will not be definitive as economic importance will vary depending on the region of the world in which a researcher is based. However, care was taken to include researchers from as many parts of the world as possible when carrying out the survey. The top 10 list emerging from the survey is composed of: (1) root-knot nematodes (Meloidogyne spp.); (2) cyst nematodes (Heterodera and Globodera spp.); (3) root lesion nematodes (Pratylenchus spp.); (4) the burrowing nematode Radopholus similis; (5) Ditylenchus dipsaci; (6) the pine wilt nematode Bursaphelenchus xylophilus; (7) the reniform nematode Rotylenchulus reniformis; (8) Xiphinema index (the only virus vector nematode to make the list); (9) Nacobbus aberrans; and (10) Aphelenchoides besseyi. The biology of each nematode (or nematode group) is reviewed briefly.

Root lesion nematodes (RLNs) are one of the most economically important groups of plant nematodes. As migratory endoparasites, their presence in roots is less obvious than infestations of sedentary endoparasites; nevertheless, in many instances, they are the major crop pests. With increasing molecular information on nematode parasitism, available data now reflect the differences and, in particular, similarities in lifestyle between migratory and sedentary endoparasites. Far from being unsophisticated compared with sedentary endoparasites, migratory endoparasites are exquisitely suited to their parasitic lifestyle. What they lack in effectors required for induction of permanent feeding sites, they make up for with their versatile host range and their ability to move and feed from new host roots and survive adverse conditions. In this review, we summarize the current molecular data available for RLNs and highlight differences and similarities in effectors and molecular mechanisms between migratory and sedentary endoparasitic nematodes.

An isolate of Japanese iris necrotic ring virus (JINRV) was detected from a plant of Iris ensata Thunb. growing in Western Australia, and the sequence of its complete genome (4019 nucleotides) was determined. The deduced replicase and coat proteins shared 75 % and 72 % amino acid identities, respectively, with those of the only other sequenced isolate of JINRV, from Japan. This is the first report of JINRV in Australia, and the first beyond Japan.

Partial genome sequences with identity to members of the genus Allexivirus were described in Australia from Allium sativum L. (garlic). Phylogenetic analysis of coat protein and nucleotide binding protein gene sequences with those of 82 other allexivirus isolates confirmed that the Australian isolates were members of Garlic virus A, Garlic virus B, Garlic virus C, and Garlic virus D.

Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust than seedling Rph (Resistance to Puccinia hordei) genes. A major gene for adult plant resistance to barley leaf rust has been mapped to the telomere region of chromosome 5HS. PCR-based molecular markers were developed for saturation of this region based on previously mapped simple sequence repeat, restriction fragment length polymorphism and Diversity Arrays Technology markers. In addition, defence gene homologue (DGH) and wheat expressed sequence tags mapped in specific bins were used to develop new PCR markers. Seventeen PCR-based markers were mapped to the short arm of chromosome 5H in 292 doubled haploid lines from a cross of Pompadour × Stirling, in which seven markers were mapped within 5 cM of the APR gene. The closest linked marker was about 0.7 cM from the APR gene. The wheat deletion bin map together with defence gene homologues was demonstrated to be an efficient tool for development of new molecular markers associated with the disease resistance gene. Four DGH markers were associated with the APR gene. The new molecular markers are a useful tool for marker-assisted selection of the APR gene and provided a better understanding of the molecular mechanism for leaf rust resistance.

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.

Thirty-two accessions of four Nicotiana species (Nicotiana benthamiana Domin, Nicotiana occidentalis H.-M.Wheeler, Nicotiana simulans N. Burb. and Nicotiana umbratica N.T.Burb.) collected from wild plants in northern Australia were assessed for responses to water stress. Under moderate water stress conditions, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, root : shoot ratio, and relative water content of leaves were significantly affected. However, the degree to which the accessions were affected varied considerably. Some accessions of N. simulans, N. benthamiana and N. occidentalis were significantly more affected by water stress than others. There was significant variation between accessions in leaf and shoot tip wilting times. Initial symptom expression (leaf wilting) was significantly delayed in three accessions of N. benthamiana, and in one accession of N. umbratica. The least water stress tolerant lines, two accessions each of N. benthamiana, N. occidentalis and N. simulans, exhibited advanced symptoms of water stress (shoot tip wilting) within 14–17 days of cessation of watering. This stage was significantly delayed in three accessions of N. benthamiana and two accessions N. occidentalis and one accession of each of N. simulans and N. umbratica, which showed tip wilting only after 21–24 days. There were variations among the accessions of same Nicotiana species on their tolerance to water stress. Plant responses to water stress could not be predicted from their plant biomass and leaf relative water content under well-watered conditions. Leaf chlorophyll content was variable under water stress, but did not correlate with water stress tolerance.

The southwest Australian floristic region (SWAFR) is an internationally recognized 'hot spot' of global biodiversity and has an endangered flora. It represents a unique interface between an ancient ecosystem and a recent agroecosystem, providing the opportunity to investigate encounters where the recipient of the virus is an introduced crop and the donor a native plant and vice versa. Phylogenetic analysis of the virus coat-protein genes was used to study isolates of three potyviruses representing different 'new encounter' scenarios at this interface. The incidence, symptomatology, host range, non-persistent aphid transmission and considerable genetic diversity of the first indigenous virus described from the SWAFR, where it infects the native legume Hardenbergia comptoniana, and its potential to damage lupin, a locally important, newly introduced cultivated grain legume, was studied. The name Hardenbergia mosaic virus is proposed for this virus. Two other examples of 'new encounter' scenarios involving other legume-infecting potyviruses studied were: Passion fruit woodiness virus, which has been found only in Australasia, where it damages recently introduced species of Passiflora and legumes; and Bean yellow mosaic virus, which is not indigenous to Australia and was introduced recently to the SWAFR, where it infects a number of introduced legumes, but also damages the local native legume Kennedia prostrata. Isolates of the former had considerable genetic diversity consistent with the virus being indigenous, while isolates of the latter virus from K. prostrata had a low genetic diversity consistent with recent arrival. This research illustrates how introduced viruses can damage indigenous plants and indigenous viruses can damage introduced cultivated plants within this unique ecosystem, and how human activities can facilitate damaging 'new encounters' between plants and viruses.

SUMMARY Gene expression studies are often carried out at the whole organism, organ or tissue levels. The different cell types present in most tissue exhibit different patterns of gene expression. This limits analyses because results obtained represent an average of the activities of the different cell types, and may lead to masking of genes of interest that are specifically expressed in a particular cell type. The recent development of laser capture microdissection (LCM) now enables target cells to be isolated from complex tissues and allows analysis of specific cell types that represent the in vivo state at the time of sample extraction. LCM has been applied to analyse plant tissues in a number of studies. This review illustrates the application of LCM in studies on gene expression profiling and proteomics, and also in research on plant-microbe interactions.

SUMMARY Root-knot nematodes (Meloidogyne spp.) are economically important plant parasites that induce specific feeding cells called giant cells in host roots. Study of molecular events involved in induction and differentiation of giant cells has been limited because it is difficult to obtain pure cytoplasm specifically from the highly specialized cells. In this work, laser capture microdissection (LCM) was used to collect cytoplasmic contents from paraffin-embedded sections of 4 day post-inoculation giant cells in tomato roots. Total RNA was isolated from the sections, and used in RT-PCR to investigate expression of cell cycle genes in giant cells. Two D-type cyclin genes, LeCycD3;2 and LeCycD3;3, were expressed at higher levels in giant cells compared with other cell-cycle-related cyclin genes, suggesting that the induction of the G1 phase of the cell cycle may be triggered in response to stimulation by the infecting nematode. LCM provides a powerful new tool to study the molecular basis of host-pathogen interactions at the cellular or subcellular level.

SUMMARY Giant cells induced by root-knot nematodes are highly specialized cells which function as transfer cells and provide nutrients to support the growth and reproduction of the nematode. Changes in the overall pattern of gene expression in giant cells occur during the formation and maintenance of the nematode feeding cells. Differential display analysis has been carried out to detect changes in gene expression in giant cells induced in tomato roots by Meloidogyne javanica, using mRNA isolated directly from mature giant cell cytoplasm, compared to non-infected root tissue. Eighty-one differential displayed bands were generated, and of these, 73 were up-regulated and 8 were down-regulated. Twenty-seven sequences were obtained by direct sequencing of the bands, and 16 fragments were further analysed by real-time quantitative RT-PCR. The most highly up-regulated transcript increased 56-fold in giant cells, and the greatest down-regulation was 11-fold. A time course of expression of the highest and lowest expressed transcripts was also undertaken by quantitative RT-PCR using giant cell enriched tissue. These showed similar changes in expression, but values were dramatically reduced. This result shows the importance of analysing giant cell cytoplasm directly, rather than starting with giant cell enriched tissue, to obtain accurate information on changes in gene expression in nematode feeding cells. Sequenced transcripts showed significant homology to mitogen-activated protein kinase, S-adenosylmethionine decarboxylase, cysteine synthase, cytochrome c reductase subunit, and ribosomal proteins. The expression analysed reflects the high metabolic rate in mature giant cells rather than processes of giant cell induction.

A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed. Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barleyMlo gene.

A plant of Iris xiphium in Western Australia displayed chlorotic streaks on the leaves and spathe that resembled symptoms of virus infection. After sequencing total RNA extracted from the plant, a sequence of 6,957 nt representing the complete genome of an Australian isolate of Narcissus mosaic virus (NMV) was obtained. It shared 97 % nucleotide identity with the two available complete genome sequences of NMV. This is the first report of natural infection of Iris by NMV.

Plants are constantly challenged by pathogens and pests, which can have a profound impact on the yield and quality of produce in agricultural systems. The vascular system of higher plants is critical for growth and for their ability to counteract changing external conditions, serving as a distribution network for water, nutrients, and photosynthates from the source organs to regions where they are in demand. Unfortunately, these features also make it an attractive target for pathogens and pests that demand access to a reliable supply of host resources. The vascular tissue of plants therefore often plays a central role in pathogen and parasite interactions. One of the more striking rearrangements of the host vascular system occurs during root-knot nematode infestation of plant roots. These sedentary endoparasites induce permanent feeding sites that are comprised of ‘giant cells’ and are subject to extensive changes in vascularization, resulting in the giant cells being encaged within a network of de novo formed xylem and phloem cells. Despite being considered critical to the function of the feeding site, the mechanisms underlying this vascularization have received surprisingly little attention when compared with the amount of research on giant cell development and function. An overview of the current knowledge on vascularization of root-knot nematode feeding sites is provided here and recent advances in our understanding of the transport mechanisms involved in nutrient delivery to these parasite-induced sinks are described.

The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction.

Septoria nodorum blotch is the most important leaf disease of wheat in Western Australia. A potentially useful source of resistance has been identified in an accession of Aegilops tauschii. To study the genetics of resistance of this source a cross was made between the resistant Ae. tauschii accession, RL5271, and a susceptible accession, CPI110889. The resistant parent took significantly longer to develop symptoms, developed significantly fewer lesions and expressed significantly lower levels of disease than the susceptible parent. The F1 mean response for disease severity indicated there was no complete dominance. The F3 families were classified using three approaches. In the first approach the individual F3 plant response was used to classify the F3 families. In the second approach the F3 family means and standard errors were used to classify the F3 families. In the final approach Best Linear Unbiased Predictors of disease score and standard error for each F3 family derived from a REML analysis were used to classify the F3 families. The genotypic ratios generated by each of the approaches suggested that resistance is controlled by a single gene. The effectiveness of the resistance and its simple genetic control in the Ae. tauschii, accession RL5271 may be a useful resistance source for use in a bread wheat breeding program.

important species because they produce essential oil in the heartwood which is used extensively in the incense A reproducible system for somatic embryogenesis and perfumery industry (Loneragan, 1990; Rao and and plantlet formation of sandalwood has been Bapat, 1995). Santalum spicatum natural stands are developed. A high frequency (100%) of somatic depleted since the rate of regeneration cannot keep pace embryos were induced directly from various explants with the rate of harvesting. Sandalwood harvesting, which in MS (Murashige and Skoog, 1962) medium with thidi- usually involves removing the entire tree, may have azuron (1 or 2 mM) or indirectly in medium containing resulted in a critical loss of genetic diversity and valuable 2,4-D plus thidiazuron. Within 8 weeks, white globular agronomic characters. Techniques for vegetative propagasomatic embryos or friable embryogenic tissue tion of WA sandalwood are not yet available. developed on cultured explants. In S. album the globu- In addition to conserving and regenerating the indigenlar somatic embryos were transferred to MS medium ous species, Indian sandalwood, which contains a higher supplemented with IAA (6 mM) and kinetin (1 mM) where oil content, is being evaluated as a potential plantation they developed further, multiplied and maintained species in the Ord river region of Western Australia friable embryogenic tissue. After 15‐30 d, mature (Applegate and McKinnell, 1993). However, S. album is somatic embryos (1‐2 mm) with well-developed cotyle- susceptible to many pests, such as fungi, nematodes (Rai, dons were separated and subcultured on to medium 1990), and spike diseases caused by micoplasma containing GA 3 (6 mM) for germination. Once germin- (Parathasarathy and Venkatesan, 1982). ated, elongated somatic embryos (10‐20 mm long) Conventional breeding of sandalwood for introgression grew further in MS supplemented with lower GA 3 of new genetic information can be an expensive and (3 mM). In S. spicatum, the addition of casein hydrolys- diYcult task because of their long generation time, sexual ate and coconut milk was necessary for plantlet devel- incompatibility and heterozygous nature (Rugkhla, opment from somatic embryos. From histological 1997). In vitro regeneration techniques can be used to studies, it appeared that primary somatic embryos clone superior lines, and are needed for Agrobacteriumarose from single cells or had a multicellular origin mediated gene transfer techniques and for protoplast from the epidermis or cortical parenchyma. Secondary fusion. Regeneration via somatic embryogenesis of S. somatic embryos and friable embryogenic tissue dif- album can be achieved from hypocotyl, nodal and endoferentiated from groups of proembryogenic cells from sperm explants (Bapat and Rao, 1979; Lakshmi Sita a superficial layer of the primary somatic embryos. et al., 1979; Rao and Bapat, 1992). Preliminary results on somatic embryogenesis of

A strong association between the absence of the granule-bound starch synthase (GBSS) protein for the 4A chromosome of wheat and Japanese Udon noodle quality has been previously described. The aim of this study was to identify a molecular marker linked to the GBSS 4A locus which could be used to identify wheat with the desired texture for Udon noodles. PCR primers were designed to target this gene which gave a 440 bp PCR band, corresponding to the presence or absence of the 4A GBSS gene. Of the 268 genotypes screened with these primers, 267 were correctly identified using the PCR primers. The remaining genotype was shown to be heterogeneous for the marker. The PCR marker test developed has advantages over existing methods used to screen for Udon noodle starch quality as it enables high throughput, accurate tests to be carried out on leaves of young seedlings or mature seed and identify breeding lines that are heterogeneous for the 4A allele which will allow for reselections. Application of this PCR test will speed up selection for Udon noodle quality genotypes and reduce breeding costs for production of noodle wheat varieties. Abbreviations: CTAB, cetyltrimethlammonium bromide; FSV, flour swelling volume; GBSS, granule-bound starch synthase; IEF, isoelectric focusing; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

Drought occurring at critical growth and developmental stages in cereals affects productivity by reducing biomass accumulation, grain set, and grain yield and quality. Maize (cv. SR-73), sorghum (cv. Trump), and wheat (cv. Spear) were established in drought-prone field conditions in Perth, Western Australia, in 1994. The plants were then subjected to optimal and suboptimal supplementary watering regimes at growth stages that were sensitive to water availability. Glycinebetaine in aqueous solution was applied to leaves at three rates (2, 4 and 6 kg ha-1 and a control) to establish whether its application could ameliorate the effects of drought on the yield of the crops. Aboveground biomass production was measured at the beginning and at termination of the watering regimes. Leaf tissue glycinebetaine concentrations were determined 1 and 3 weeks after application. At physiological maturity, grains from the crops were harvested and grain yield, number of grains m-2 and single grain weight were recorded. Drought significantly reduced above-ground biomass production in maize (P = 0.047), but not in sorghum and wheat. Grain yield of maize, number of grains m-2 of maize and sorghum, and single grain weight of sorghum were significantly depressed by drought. Foliar application of aqueous glycinebetaine marginally enhanced biomass production in the three crops and significantly increased grain yield of maize (P = 0.001) and sorghum (P = 0.003). It also resulted in more grains m-2 of maize, sorghum and wheat (P = 0.001, 0.001 and 0.003, respectively), with interactions between water and glycinebetaine treatments for sorghum and wheat (P = 0.001 and 0.001, respectively). Residual tissue glycinebetaine levels remained high 3 weeks after application to the crops. The positive effects of glycinebetaine treatment appear to be linked to its physiological role as a plant osmoticum that improves drought tolerance. The results of these studies suggest that foliar application of glycinebetaine may be used to improve drought tolerance and economic yield of maize and sorghum, but not of wheat. Increased grain yield was associated with more grains m-2 rather than greater single grain weight.

* In this study, we tested whether the organogenesis of symbiotic root nodules, lateral roots and root galls induced by parasitic root knot nematodes (Meloidogyne javanica) was regulated by the presence of flavonoids in the roots of Medicago truncatula. Flavonoids accumulate in all three types of root organ, and have been hypothesized previously to be required for secondary root organogenesis because of their potential role as auxin transport regulators. * Using RNA interference to silence the flavonoid biosynthetic pathway in M. truncatula, we generated transformed flavonoid-deficient hairy roots which were used to study flavonoid accumulation, cell division and organogenesis of nodules, lateral roots and root galls. * Flavonoid-deficient roots did not form nodules, as demonstrated previously, but showed altered root growth in response to rhizobia. By contrast, flavonoid-deficient roots showed no difference in the number of lateral roots and root galls. Galls on flavonoid-deficient roots formed normal giant cells, but were shorter, and were characterized by reduced numbers of dividing pericycle cells. * We rejected the hypothesis that flavonoids are required as general regulators of the organogenesis of secondary root organs, but flavonoids appear to be necessary for nodulation. Possible reasons for this difference in the requirement for flavonoids are discussed.

Starchy endosperm protoplasts from developing barley (Hordeum vulgare) grains were isolated 8–15 days post-anthesis. These endosperm protoplasts were incubated in a hormone free medium containing sucrose as a carbon source and glutamine as a nitrogen source. In this medium the endosperm protoplasts remained viable for several days and starch synthesis was also observed. Transient expression of chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) reporter genes linked to the CaMV 35S promoter was detected 18–20 h after PEG induced DNA uptake. GUS fusions to two endosperm specific promoters, a wheat high molecular weight (HMW) glutenin and a barley chymotrypsin-inhibitor 2 were also functional in the endosperm protoplasts. Similarly, GUS activity could be detected when either the CaMV 35S or HMW glutenin promoters linked to the GUS gene were introduced into intact endosperm tissues of 15–20 days post-anthesis barley grains by microprojectile bombardment.

It has generally been assumed that plants micropropagated by multiplication of organised meristems or by serial subculture of stem nodes are genetically stable. This assumption has been tested for potato plants, all of which originated from one shoot culture of cv Desiree, that have been propagated or stored under different regimes in vitro, by two methods. The first is by developing DNA restriction fragment analysis and using it to study changes in DNA banding patterns of regenerated plants, and the second is morphological analysis of the same plants grown in the field. Potato plants regenerated via callus can exhibit somaclonal variation, and so plants regenerated via a brief callus phase from the same stock plant of Desiree were also analysed. Molecular analysis involved combinations of 4 single or low copy DNA probes and 4 restriction enzymes. Variation in DNA fragment patterns was not found in any of the 128 plants micropropagated through 6 rounds of nodal subculture, nor in 18 plants that had been stored at low temperature or in 21 plants stored by osmotic reduction of growth. In contrast, 6 of 46 plants regenerated from leaf callus exhibited variations in banding pattern. In the morphological analysis, variants were also only identified in the population of plants regenerated via leaf callus. These results support the concensus view that the plants obtained from micropropagation of meristems are stable genetically, in contrast to those regenerated via callus. However, with the combinations of DNA probes and restriction enzymes used in the analysis, it is not possible to state that variation was not present. Restriction fragment analysis is a suitable approach for analysis of genetic stability of stock plants in vitro, but more plants can be screened for stability by morphological analysis in the field if time delays of 1–2 years are acceptable.

The root-knot nematode Meloidogyne javanica induces giant cells and feeds from them during its development and reproduction. To study the cellular processes underlying the formation of giant cells, laser microdissection was used to isolate the contents of early-stage giant cells 4 and 7 days post-infection (dpi) from tomato, and cDNA libraries from both stages were generated with 87 [250 expressed sequence tag (EST) clones] and 54 (309 EST clones) individual transcripts identified, respectively. These transcripts have roles in metabolism, stress response, protein synthesis, cell division and morphogenesis, transport, signal transduction, protein modification and fate, and regulation of cellular processes. The expression of 25 selected transcripts was studied further by real-time quantitative reverse transcriptase-polymerase chain reaction. Among them, 13 showed continuous up-regulation in giant cells from 4 to 7 dpi. The expression of two transcripts was higher than in controls at 4 dpi and remained at the same level at 7 dpi; a further five transcripts were highly expressed only at 7 dpi. The Phi-1 protein gene, a cell cycle-related homologue in tobacco, was expressed 8.5 times more strongly in giant cells than in control cells at 4 dpi, but was reduced to 6.7 times at 7 dpi. Using in situ hybridization, the expression of the Phi-1 gene was preferentially localized in the cytoplasm of giant cells at 4 dpi, together with a pectinesterase U1 precursor gene. The identification of highly expressed transcripts in developing giant cells adds to the knowledge of the plant genes responsive to nematode infection, and may provide candidate genes for nematode control strategies.

By measuring uptake of the membrane impermeable dye. phenosafranine, it can be shown that the plasma membrane of intact cells within cell aggregates can be reversibly permeabilized by electroporation. However, the plant cell wall is a barrier to DNA uptake by intact cells, although under certain circumstances expression of DNA, electroporated into intact cells, can be demonstrated. The level of expression is about 20–50 times lower than that obtained by electroporation of protoplasts, and depends on cell wall properties and pretreatments of cell aggregates. In contrast, efficient transformation of whole cells of bacteria and yeasts can be achieved by electroporation. Factors which influence DNA transfer into whole plant cells and the possibility of stable transformation are discussed.

Amongst tetrapoid somatic hybrids of Solanum tuberosum and Solanum brevidens, the expression of resistance to potato virus X (PVX), potato virus Y (PVY) and potato leaf roll virus (PLRV) ranged between that of the fusion parents. Genotypes with general morphology resembling S. brevidens generally were more resistant to virus infection. In contrast, hexaploid hybrids could be divided into high and low resistance groups, resistance being associated with two doses of the S. brevidens nuclear genome, but again genotypes with general morphology resembling S. brevidens were more resistance. Correlation of resistance to the different viruses was observed in aneuploid hybrids lacking parental chromosomes. This observation suggests that resistance/susceptibility is associated with the loss of individual S. brevidens or S. tuberosum chromosomes.

The root-knot nematodeMeloidogyne javanica was identified in papaya roots in Western Australia. Species identification was based on esterase isoenzyme patterns and PCR diagnostics.