Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Soil temperatures and inoculation techniques affect emergence and reisolation of Sclerotinia sclerotiorum from soybean

Emergence of Amsoy soybean (Glycine max) seed inoculated withSclerotinia sclerotiorum was significantly reduced below noninoculated seed at soil temperatures of 25°, 30°, and 35 °C, but not at 20 °C.S. sclerotiorum was readily reisolated from wound-inoculated stems of seedlings and nearly mature plants above the point of inoculation and below to the crown area, but not from roots. The fungus was recovered from stems but not roots of 15-day seedlings grown in sterile soil before infestation of the soil surface with a suspension of mycelium and sclerotia and assayed at 15 days after soil infestation. When compared to healthy, seeds, infected seeds withS. sclerotiorum were characterized by appearing flattened.Supported in part by the Illinois Agricultural Experiment Station; Regional Project S-72; and U.S. Agency for International Development, grant csd-1922.     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Treatment of lentil (Lens culinaris. L) seeds with various concentrations of salts to check their efficiency against seed-borne mycoflora during storage

Seed sample of lentil collected from the Swabi district were treated with NaCl and KCl at 0.01, 0.1 and 1.0% (w/w). Seed-borne mycoflora was observed at 0, 20, 40, 60 and 80 day intervals. Seed treatment with both the salts was found to be effective against storage fungi; however, KCl was more efficient against storage mycoflora such as Aspergillus species when compared with NaCl. With the passage of time, the incidence of deep-seated fungi was observed in salt-treated seed samples while untreated seed sample showed heavy infestation by the species of Aspergillus. Seed viability also remained unaffected in storage, except in seeds heavily infested with seed-borne mycoflora. Aspergillus spp. were the main cause of seed rot. Surface sterilisation of seeds with 1% Na(OCl)₂ reduced the fungal infestation of seeds. Among various concentrations of salts, 0.1% (w/w) of both salts were better in controlling seed-borne mycoflora.     

Yield,development, and quality response of dual‐toxin Bt cotton to Helicoverpa spp. infestations in Australia

To verify current thresholds for Bollgard II^® cotton in Australia, the impact of Helicoverpa spp. (Lepidoptera: Noctuidae) larvae on yield, development, and quality under various infestation intensities and durations, and stages of growth, was tested using small plot field experiments over two seasons. Infestation with up to 80 eggs m^?1 of Helicoverpa armigera (Hübner) and Helicoverpa punctigera Wallengren showed that species, infestation level, and stage of growth had no significant effect on yields of seed‐cotton or lint and on maturity and fibre quality. The duration of infestation of white flowers with H. punctigera neonates (maximum of every day for up to 4 weeks) had no impact on the yield of seed‐cotton or lint, maturity, and fibre quality, but when 100% of flowers were infested (compared with 0 or 50%), seed‐cotton and lint yields were significantly reduced and maturity was delayed. Infestation with up to 18 medium H. armigera larvae m^?1 at several plant stages did not significantly affect yields of seed‐cotton and lint, maturity, and fibre quality. A heliocide spray applied on a commercial farm at the current threshold resulted in a significantly higher lint yield, compared with a farm where no spray was applied. In conclusion, Bollgard II^® cotton is highly resistant to Helicoverpa spp. infestation.     

Induction of Resistance in Melon to Didymella bryoniae and Sclerotinia sclerotiorum by Seed Treatments with Acibenzolar-S-methyl and Methyl Jasmonate but not with Salicylic Acid

The plant resistance activator acibenzolar‐S‐methyl (BTH), the signalling molecules salicylic acid (SA) and methyl jasmonate (MeJA) were tested by seed treatment for their ability to protect melon seedlings from gummy stem blight and white mould disease caused by the soil‐borne fungal pathogens Didymella bryoniae and Sclerotinia sclerotiorum, respectively. Didymella bryoniae infection on melon seedlings was completely suppressed by MeJA treatment. Necrotic lesions akin hypersensitive response occurred on all inoculated seedlings and prevented pathogen diffusion into healthy tissues. Didymella bryoniae infection was restricted following BTH seed treatment as well, although the percentage of necrotic lesions in comparison with the water soaked lesions was significantly lower than that from MeJA‐induced seedlings. BTH protected melon seedlings against S. sclerotiorum by the occurrence of a high percentage of necrotic lesions. A lower level of resistance was also achieved by MeJA seed treatment. The augmented level of resistance of tissues from BTH and MeJA‐treated seeds was associated with rapid increases in the activity of the pathogenesis‐related proteins chitinase and peroxidase. MeJA also determined a rapid and transient accumulation of lipoxygenase. Moreover, BTH and MeJA treatments determined the differential induction of particular de novo synthesized isoenzymes of these proteins. Results indicate that BTH and MeJA applied to melon seeds may activate on seedlings diverse metabolic pathways leading to the enhancement of resistance against distinct pathogens.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

PCR-based detection of Phomopsis azadirachtae in die-back affected neem seeds

Abstract

Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories.     

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β‐N‐oxalyl‐L ‐α,β‐diaminopropionic acid (β‐ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed‐specific expression of an oxalate‐degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β‐ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC‐expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate‐degrading enzyme.     

Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and fleas

Arthropod‐borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod‐borne pathogens via quantitative real‐time (qPCR), or even hand‐held detection devices, a simple and robust maceration and nucleic acid extraction method is necessary to implement biosurveillance capabilities. In this study, a comparison of maceration techniques using five types of beads followed by nucleic acid extraction and detection were tested using two morphologically disparate arthropods, the Aedes aegypti mosquito and Xenopsylla spp. flea, to detect the zoonotic diseases dengue virus serotype‐1 and Yersinia pestis. Post‐maceration nucleic acid extraction was carried out using the 1‐2‐3 Platinum‐Path‐Sample‐Purification (PPSP) kit followed by qPCR detection using the Joint Biological Agent Identification and Diagnostic System (JBAIDS). We found that the 5mm stainless steel beads added to the beads provided in the PPSP kit were successful in macerating the exoskeleton for both Ae. aegypti and Xenopsylla spp. Replicates in the maceration/extraction/detection protocol were increased in a stepwise fashion until a final 128 replicates were obtained. For dengue virus detection there was a 99% positivity rate and for Y. pestis detection there was a 95% positive detection rate. In the examination of both pathogens, there were no significant differences between qPCR instruments, days ran, time of day ran, or operators.     

Quantitative real‐time PCR detection of a harmful unarmoured dinoflagellate,Karlodinium australe (Dinophyceae)

We investigated a harmful algal bloom (HAB) associated with the massive fish kills in Johor Strait, Malaysia, which recurred a year after the first incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green‐based real‐time PCR (qPCR) to detect the culpable dinoflagellate species, Karlodinium australe. Species‐specific qPCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (rDNA). The species specificity of the primers designed was evaluated by screening on the non‐target species (Karlodinium veneficum, Takayama spp., and Karenia spp.) and no cross‐detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 ± 505 (P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically (R² = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single‐cell PCR and rDNA sequencing from the field samples further confirmed the finding. With the sensitivity as low as five cells, the qPCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose.     

Rapid and sensitive detection of Acidovorax citrulli in cucurbit seeds by visual loop‐mediated isothermal amplification assay

A rapid, sensitive and visual loop‐mediated isothermal amplification (LAMP) method for detecting Acidovorax citrulli in cucurbit seed was developed in this study. The LAMP primers were designed to recognize the non‐ribosomal peptide synthetase (NRPS) gene (locus tag: Aave_4658) from A. citrulli. The LAMP assay was conducted at 64°C in 1 hr with calcein as an indicator. The sensitivity and specificity of the LAMP assay were further compared with those of a conventional polymerase chain reaction (PCR). The LAMP assay is highly specific to A. citrulli, and no cross‐reaction was observed with other bacterial pathogen. The sensitivity of the LAMP assay was 100‐fold higher than that of conventional PCR with a detection limit of 1 pg of genomic DNA. Using the LAMP assay, 7 of 12 cantaloupe seedlots collected from Xinjiang province were determined to be positive for A. citrulli. In contrast, only 2 of 12 seedlots showed positive for the pathogen with conventional PCR. Moreover, A. citrulli was detected in 100% of artificially infested seedlots with 0.01% infestation or greater. Our results demonstrated that the LAMP assay was simple, visual and sensitive for detecting A. citrulli, especially in seed health testing. Hence, this method has great potential application in routine detecting seed‐borne pathogens and reducing the risk of epidemics.     

Soil temperatures and inoculation techniques affect emergence and reisolation of sclerotinia Sclerotiorum from soybean

Emergence of Amsoy soybean (Glycine max) seed inoculated withSclerotinia sclerotiorum was significantly reduced below noninoculated seed at soil temperatures of 25, 30 and 35 °C, but not at 20 °C.S. sclerotiorum was readily·reisolated from wound-inoculated stems of seedlings and nearly mature plants above the point of inoculation below to the crown area, but not from roots. The fungus was recovered from stems but not roots of seedlings grown in sterile soil for 15 days before infestation of the soil surface with a suspension of mycelium and sclerotia and assayed at 15 days after soil infestation. When compared to healthy, seed infected withS. sclerotiorum were characterized by appearing flattened.Supported in part by the Illinois Agricultural Experiment Station; Regional Project S-72; and U.S. Agency for International Development, grant csd-1922.     

Sowing seasons and drying methods during post-harvest influence the seed vigour of soybean (Glycine max (L.) Merr.)

Experiments were conducted to study the influence of sowing seasons and drying methods on the seed vigour of two spring soybean (Glycine max (L.) Merr.) cultivars. Two cultivars, ‘Huachun18’ and ‘Huachun 14’, were sown in three seasons viz., spring, summer and autumn and the harvested seeds were dried using three different methods. The results showed that soybean sown in spring had a higher number of branches per plant, pods per branch and seed weight, and consequently resulted in higher seed yields than that of soybean sown in autumn or summer seasons. Seeds sown in the autumn season had the lowest values of electrical conductivity during seed imbibitions, higher peroxidase (POD) activity in germinated seedlings and lower contamination by the seed-borne fungi on the MS medium, which indirectly improved the seed vigour, which was followed by summer sown seeds. Seeds sown during the spring season resulted in poor seed vigour. In addition, the effect of drying methods on the seed vigour was also clarified. Seeds that hung for four days before threshing and then air-dried had the poorest seed vigour which was determined by germination, electrical conductivity, POD activity and seed borne fungal growth. There was no difference in seed vigour between other methods, i.e. seeds threshed directly at harvest and then air-dried on a bamboo sifter or concrete floor. These results indicated that autumn sowing soybean and the drying method in which seeds were threshed directly at harvest and then air-dried on a bamboo sifter resulted in higher seed vigour.     

Non‐pollinating Fig Wasps Decrease Pollinator and Seed Production in Ficus andicola (Moraceae)

Fig trees ( Ficus spp.) and Agaonine fig‐wasps participate in an obligate mutualism. Fig wasps can only develop within fig inflorescences (syconia) and they are the only organisms capable of pollinating fig flowers. Other non‐pollinating wasps that lay eggs by inserting their ovipositors from the outside can also develop in syconia. These parasitic wasps may be parasitoids of either pollinating or other non‐pollinating wasps, or form galls in fig flowers or other tissues. Depending on this interaction, parasitic wasps may have various effects on the production of pollinating wasps and seeds. Wasps in the genus Idarnes, which parasitize New World figs (subgenus Urostigma), have an effect on wasp production but not on seed production. Heterandrium spp., which have short ovipositors and lay on external flowers, are infrequent and no effect on seed production has been documented. In the Colombian Andes, Idarnes spp. and Heterandrium spp. are the most frequent parasites of the Ficus andicola — Pegoscapus sp. mutualism, affecting 62 and 43 percent of syconia, respectively. Controlling for other factors that influence wasp and seed production, such as number of foundresses, syconium size and tree, we found that Idarnes reduced pollinator production by almost half but did not reduce seed production, whereas Heterandrium reduced seed production by 40 percent, and marginally affected pollinator production. Our results provide the first clear documentation of Heterandrium spp. impact on fig seed production. Whether the relative abundance of this genus is a generalized phenomenon in montane forest remains to be determined.     

Effect of Herbicides and Oat Amendment on Soybean Disease Development and Seed Quality

The effects of four herbicides and hand weeding and no weeding on soybean disease development and seed quality were studied in the field. Chloramben, dinoseb, fluchloralin, and trifluralin were preplant incorporated at recommended rates. In addition, the effect of an in-furrow oat (Avena sativa L.) amendment used as an inoculum carrier was studied in all treatments. Compared with the hand-weeded control, yield, incidence of Septoria leafspot (Septoria glycines), and the recovery of Phomopsis spp. from pod and stem tissues were reduced in the week-infested control and dinosebtreated plots. Recovery of Alternaria spp. was greater from crown tissues of plants grown in chloramben- and fluchloralin-treated plots compared with, either the weed-infested or hand-weeded control. Septoria leafspot ratings and recovery of Phomopsis spp. from various plant tissues were higher in plants from hand-weeded plots compared with weed-infested plots, whereas the opposite was truefor, the recovery of Fusarium spp. Chloramben-treated plots were high-yielding and had the best weed control of all herbicide treatments. Seed quality was not affected by herbicide treatment. Plants from furrows with autoclaved oats had a lower incidence of Septoria glycines, a greater recovery of Fusarium spp. from pod and stem pieces, and produced seed with lower quality than plants from rows with no oat amendment.     

Phomopsis (Diaporthe) Species as the Cause of Soybean Seed Decay in Korea

Soybeans are an important crop known to harbour a complex of Diaporthe and Phomopsis species. This complex has been reported to be involved in several soybean diseases, including Phomopsis seed decay. In this study, two species of Diaporthe/Phomopsis fungi from soybean plants were identified by morphological and molecular characterizations. Koch's postulates were confirmed by pathogenicity tests on hypocotyls of soybean seedlings. Phomopsis longicolla was found to be the most common and virulent pathogen to soybeans in Korea. Phomopsis sp., which was considered as a new soybean pathogen, might have been introduced from other plants given that similar strains of Phomopsis sp. have infected fruit trees in China, Japan and Portugal and vegetable plants in the United States.