The presence of double-stranded RNAs in <Emphasis Type="Italic">Alternaria alternata</Emphasis> Japanese pear pathotype is associated with morphological changes

Strains of the Japanese pear pathotype of Alternaria alternata were screened for double-stranded RNAs (dsRNAs). Four strains had several dsRNAs; strain N18 was associated with several dsRNAs and had impaired growth phenotypes such as irregular mycelium and abnormal pigmentation. We isolated dsRNA-cured isolates from strain N18 by single-conidium isolation. The dsRNA-cured isolates had recovered normal growth and pigmentation. Enlarged vesicles were observed in mycelial cells of the original dsRNA-carrying N18 strain. DAPI nuclear staining revealed regression of the nuclei in dsRNA-carrying N18 cells. These results indicate that the dsRNAs might have negative effects, such as apoptosis-like cell death, on the host fungus.     

A molecular mechanism of resistance to streptomycin in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzicola</Emphasis>

Xanthomonas oryzae pv. oryzicola, the causal agent of rice leaf streak disease, was found to be sensitive to streptomycin (an aminocyclitol glycoside antibiotic), by inhibition of protein synthesis resulting from interference with translational proofreading. This study aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzicola to streptomycin. Seven streptomycin-resistant mutants were obtained by UV induction or streptomycin selection. These mutants can grow at 100 μg ml⁻¹ of streptomycin while the wild-type strain (RS105) cannot grow at 5 μg ml⁻¹. Sequencing indicated that the rpsL gene encoding ribosomal protein S12 has 375 bp encoding 125 amino acid residues. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS and pUFRRX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. Both transformants, RS1 and RS2, could grow in the medium containing 50 μg ml⁻¹ of streptomycin. A mutation at codon 43 or 88 in rpsL can result in resistance of Xanthomonas oryzae pv. oryzicola to streptomycin.     

Progesterone supplementation during multiple ovulation treatment in buffalo species (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The aim of this study was to evaluate the effect of exogenous progesterone supplementation on superovulatory response in buffaloes that has undergone a multiple ovulation program. Fourteen Mediterranean buffaloes were divided into two groups and received a 4-day decreasing dosage of an equal mixture of 500 IU of FSH and LH starting on day 8 of the cycle. In group A (n = 7) a progesterone-releasing intravaginal device was removed on day 8, whereas in group B (n = 7) it was left till day 10, when PGF_2a {\hbox{PG}}{{\hbox{F}}_{{2}\alpha }} was administered. Eighty hours later, buffaloes were artificially inseminated and after 6 days they undergone uterine flushing. A higher (P < 0.05) number of corpora lutea (8.3 vs. 5.7) and embryo/flushing/buffalo (2.3 vs. 1.3) were recorded in group B vs. group A if responsive buffaloes are considered (n = 12) and the number of corpora lutea was highly correlated with the number of embryos (r = 0.65; P < 0.05). In conclusion, progesterone supplementation during the first 2 days of the superovulation treatment seems to enhance the recovery rate in buffalo species. A high ovulation rate, associated with a high number of corpora lutea, can represent a parameter for estimating embryo recovery.     

Temporal changes in endogenous estrogens and expression of behaviors associated with estrus during the periovulaory period in Murrah buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The objective of this study were (1) to establish the duration of behavioral estrus signs and timing of ovulation in Murrah buffaloes (n = 10) and (2) to determine relationship between behavioral estrus signs with change in plasma estrogen concentrations. Estrus and its behavioral signs were detected at hourly intervals by visual observations, per recta examination of genitalia and bull parading four times in a day for 30 minutes each. Among the behavioral signs of estrus, swollen vulva (80%) was the best indicator of estrus followed by excitement (70%). Among the duration of behavioral estrus signs the first and longest duration of estrus signs was swollen vulva which was seen upto 19.8 ± 0.8 h after onset of estrus. The mean total duration of estrus symptoms from appearance to disappearance of all the behavioral estrus symptoms was 23.5 ± 1.7 h. All the behavioral estrus symptoms were observed during the period of estrogen surge. Endocrine profile during the periestrus period showed that the mean peak concentrations of total estrogen 23.9 ± 3.9 pg/ml occurred at 8.8 ± 1.7 h after onset of estrus. The average number of estrus symptoms observed per animal during onset of spontaneous estrus was 5.7. Ovulation occurred after 37.4 ± 1.7 h after onset of estrus and 13.4 ± 1.0 h after end of total estrogen surge respectively. In conclusion, our results suggest that all signs of behavioral estrus occurred during the preovulatory rise in estrogens. The first sign of estrus to be observed was a swollen vulva and this symptom persisted the longest.     

Immunohistochemical characterization of neurons in the porcine ciliary ganglion

The present study was aimed at disclosing the chemical coding of nerve structures in the porcine ciliary ganglion (CG) using immunohistochemical methods. The substances under investigation included markers of "classical" neurotransmitters, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DbetaH) as well as neuropeptides, somatostatin (SOM), galanin (GAL), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Immunoreactivity to ChAT and VAChT was found virtually in all the neuronal somata and in numerous intraganglionic, varicose nerve fibres which often formed basket-like formations around the nerve cell bodies. Many CG neurons contained immunoreactivity for SOM (46%) or GAL (29%). Interestingly, a small number (approx. 1%) of the cholinergic somata stained for TH but not for DbetaH; nevertheless, some extra- and intraganglionic nerve fibres displayed immunoreactivity for DbetaH or TH. The CG perikarya stained neither for vasoactive intestinal polypeptide (VIP) nor for neuropeptide Y (NPY), but some NPY- or VIP-positive nerve terminals were observed within nerve bundles distributed outside the ganglion. SP- and CGRP-immunoreactivity was found in some intraganglionic nerve fibres only. The present study revealed that the porcine CG consists of cholinergic neurons many of which contain SOM and GAL. Thus, it can be assumed that in the pig, these neuropeptides are involved, complementary to acetylocholine, in the parasympathetic postganglionic nerve pathway to structures of the eye including the ciliary and iris sphincter muscles.     

AlgU contributes to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis> DC3000 by regulating production of the phytotoxin coronatine

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen to investigate the molecular basis of plant–pathogen interactions. The function of many potential virulence factors encoded in the Pst DC3000 genome and their modes of action are not fully understood. P. syringae is known to produce the exopolysaccharide alginate. Although AlgU, a sigma factor, is known to regulate the expression of genes such as algD related to alginate biosynthesis, the molecular mechanisms of AlgU in the virulence of Pst DC3000 is still unclear. To investigate the function of AlgU and alginate in plant–bacterial pathogen interactions, we generated ΔalgU and ΔalgD mutants. After inoculation with ΔalgU but not ΔalgD, host plants of Pst DC3000 including tomato and Arabidopsis had milder disease symptoms and reduced bacterial populations. Expression profiles of Pst DC3000 genes revealed that AlgU can regulate not only the expression of genes encoding alginate biosynthesis, but also the expression of genes related to type III effectors and the phytotoxin coronatine (COR). We also demonstrated that the ΔalgU mutant showed full virulence in the Arabidopsis fls2 efr1 double mutant, which is compromised in the recognition of PAMPs. Further, the application of COR was able to restore the phenotype of the ΔalgU mutant in the stomatal response. These results suggest that AlgU has an important role in the virulence of Pst DC3000 by regulating COR production.     

Effect of <Emphasis Type="Italic">Metarhizium guizhouense</Emphasis> infection on mating competition and mate choice of <Emphasis Type="Italic">Bactrocera latifrons</Emphasis> (Diptera: Tephritidae)

Metarhizium guizhouense PSUM02 treated males of Bactrocera latifrons were investigated for the mating competition among males and mating choice by female flies to develop an auto-dissemination for the control of B. latifrons. In the present study, on day 1–4 of experiment, M. guizhouense–treated male flies were equally competitive with the normal male flies as we did not observe any differences in mating by treated and normal male flies of B. latifrons. Further, mating competitiveness were found low in treated adult male B. latifrons than normal male B. latifrons from 5th days of treatment until death. Kaplan-Meier survival analysis of treated male flies gave average survival times (AST) of 4.3?±?0.1 days, while the healthy female and male flies in the same cage showed AST of 9.3?±?0.3 and 8.3?±?0.4 days, respectively. The AST of untreated flies in control experiment ranged from 14.2–14.5 days. In mating preference experiment, M. guizhouense–treated male flies were chosen by virgin female than gravid female flies for mating. The treated male flies caused mortality in both virgin and gravid female flies in the same cage with AST of 4.4?±?0.1, 5.6?±?0.1 and 7.4?±?0.2 days, respectively, while untreated flies showed AST ranged from 13.9–14.3 days in control. The treated male flies could transmit the fungus infection to both untreated female and male flies as well as in virgin and gravid female flies by mating and contact. Our experiments showed the potentiality of M. guizhouense PSUM02 in management of B. latifrons by auto-dissemination with treated male flies, which transmit the fungus to a healthy population to reduce insect pest infestations.     

Feeding potential of <Emphasis Type="Italic">Cryptolaemus montrouzieri</Emphasis> against the mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis>

Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is an exotic species native to the USA, damaging cotton and other plant families. The feeding potential of different development stages of Cryptolaemus montrouzieri Mulsant, a biological control agent against mealybugs, was investigated on different development stages of P. solenopsis. Fourth instar grubs and adults of C. montrouzieri were the most voracious feeders on different instars of mealybug. The number of 1^st instar nymphs of mealybug consumed by 1^st, 2^nd, 3^rd and 4^th instar larvae and adult beetles of C. montrouzieri was 15.56, 41.01, 125.38, 162.69 and 1613.81, respectively. The respective numbers of 2^nd and 3^rd instar nymphs of mealybug consumed were 11.15 and 1.80, 26.35 and 6.36, 73.66 and 13.32, 76.04 and 21.16, 787.95 and 114.66. The corresponding figures for adult female mealybugs were 0.94, 3.23, 8.47, 12.71 and 73.40, respectively. The results indicate that C. montrouzieri has the potential to be exploited as a biocontrol agent in North India; inoculative releases of 4^th instar larvae and adults may provide instant control of P. solenopsis. Field experiments should be conducted to determine the efficiency of the ladybird on this mealybug.